Artificial RNA for regulating RNA fragments

Abstract

We provide artificial circular RNA. [Solution] An artificial circular RNA suitable for disrupting one or more targeted disruption structures of one or more RNA fragments by hybridization, wherein the artificial circular RNA comprises 150 to 800 nucleotides, comprises two or more hybridization regions, the one or more targeted disruption structures comprises at least a hairpin loop before or after a region of unpaired nucleotides, and comprises a target hybridization region before or after a double-stranded region of at least 5 nucleotides, and the at least one target hybridization region completely hybridizes with each of the two or more hybridization regions of the artificial circular RNA, thereby disrupting the targeted disruption structure.

Description

[Technical Field]

[0001] This invention relates to the field of biotechnology. In particular, this invention relates to artificial RNA as defined herein. More specifically, this invention relates to artificial RNA suitable for modulating one or more RNA fragments by disrupting one or more targeted disruption structures of one or more RNA fragments by hybridization. [Background technology]

[0002] A vast amount of research and development is underway to expand the range of drugs in general, including antiviral drugs for treating pathogens not currently on the market, or for treating cancer. Furthermore, as viruses and tumor cells tend to mutate and become drug-resistant, what is needed now is to develop an antitumor and / or antiviral drug pipeline and refresh it with new therapies. According to a recent market report (https: / / www.mordorintelligence.com / industry-reports / global-antiviral-drugs-market-industry), the opportunities available in this market are (i) the growing need for broad-spectrum antiviral drugs, and (ii) access to antiviral drugs in the pharmaceutical market.

[0003] Currently approved effective antiviral therapies include molecules that specifically interact with viral proteins essential to the viral lifecycle. Developing these therapeutic molecules is costly and time-consuming. Furthermore, all molecules function only against specific viruses. Several efforts have been made to develop RNA-based antiviral therapies targeting viral RNA genomes [Brice A Sullenger and Smita Nair. From the RNA world to the clinic. Science 352(6292), 1417-1420]. These include the design of 19-nucleotide microRNAs (miRNAs) that specifically interact with viral RNA genomes, leading to their subsequent degradation by the cellular RISC system. Despite success in cell culture, a major drawback for their clinical use is the rapid emergence and selection of mutations in the RNA genome that inhibit interaction with miRNAs. Another drawback of miRNAs as antiviral molecules is their stability, as they are spontaneously degraded by cell disintegration mechanisms.

[0004] RNA-based therapies have gained significant attention in recent years due to the cost and time required to develop new therapeutic agents against viral proteins. RNA-based therapies have been able to address targets that cannot be treated with antibody and small molecule approaches [Ling-Ling Chen. The biogenesis and emerging roles of circular RNAs. Nature Reviews Molecular Cell Biology 17,205-211(2016),doi:10.1038 / nrm.2015.32]. As a result, multiple companies have emerged to develop RNA-based therapies to treat multiple diseases. For example, Moderna is developing an mRNA-based vaccine against infection caused by SARS-CoV-2 (Covid-19). These companies are focusing on the use of antisense, siRNA, aptamers, and microRNA mimetics / anti-microRNAs. However, these molecules (i) degrade rapidly and (ii) share the emergence of drug resistance due to the virus's high tendency to mutate, as with protein and antibody-based therapies.

[0005] The inventors of this invention focused particularly on stable artificial RNA such as circular RNA. Circular RNA is, Circular RNAs are backsplicing products of naturally occurring precursor mRNA within cells (see review article [Ling-Ling Chen. The biogenesis and emerging roles of circular RNAs. Nature Reviews Molecular Cell Biology 17, 205-211 (2016), doi:10.1038 / nrm.2015.32]). Previously considered unrelated byproducts, they have now been shown to perform several functions, including miRNA sponges and RBP (RNA-binding protein) sponges. Circular RNAs have no ends. This is crucial because most intracellular mRNA degradation pathways require cellular exonucleases to perform their degradation functions using either the 5' or 3' end. As a result, circRNAs are extremely stable molecules. The potential of circRNAs as a novel therapeutic platform is not yet fully utilized.

[0006] International Publication No. 2017 / 222911 discloses the use of circular RNA generated with exogenous introns to stimulate an immune response, or circular RNA generated with endogenous introns to prevent immune recognition of foreign RNA.

[0007] International Publication No. 0061595 discloses a covalently-closed multiple antisense (CMAS) oligo constructed to form a closed structure by ligation using complementary primers, and a ribbon-type antisense (RiAS) oligo comprising two loops containing multiple antisense sequences and a stem connecting the two loops, which are constructed by ligation using complementary sequences at both 5-prime ends.

[0008] International Publication No. 2013 / 162350 relates to the use of a circular RNA composed of two purine-rich domains that can target the pyrimidine-rich region of a virus in order to form a triple helix that can inhibit viral replication.

[0009] Chinese Patent Application Publication No. 108165549 relates to the use of circular RNA as a microRNA sponge. This presents a synthetic formulation of the well-known function of endogenous circRNA in which multiple partially complementary regions target mature microRNAs that activate the AGO2 pathway during hybridization.

[0010] Furthermore, there is a growing need to better understand the molecular mechanisms underlying many pathological conditions, including cancer, viral infections, autoimmune diseases, neurological disorders, and genetic disorders. RNA plays a role in the underlying mechanisms of most of these conditions. Therefore, there is a need for tools that enable the study of specific functions of RNA involved in the molecular mechanisms contributing to these pathological conditions.

[0011] This invention addresses the above-mentioned problems and relates to a tool that enables the regulation of the functionality of RNA fragments, such as viral genomes, enabling multiple applications. For example, the tool of the present invention (artificial RNA) can be used to study the structure-function relationship of a specific RNA fragment or a specific region within a specific RNA fragment. In addition, the RNA of the present invention can be used to prevent and / or treat diseases involving RNA fragments, such as viral infections, cancer, immune diseases, and genetic disorders. For example, the artificial RNA of the present invention can be used to study RBP (ribosome-binding protein) that binds to double-stranded RNA motifs. [Overview of the Initiative]

[0012] In a first aspect, the present invention provides an artificial circular RNA suitable for disrupting one or more target disruption structures of one or more RNA fragments by hybridization, (a) The artificial circular RNA contains 150 to 800 nucleotides, preferably 200 to 600 nucleotides. (b) The artificial circular RNA comprises two or more hybridization regions, (i) Completely hybridize with at least one target hybridization region contained in one or more target disruption structures of one or more RNA fragments, (ii) Having a total of 7 to 100 nucleotides, preferably 10 to 50 nucleotides, (c) One or more target destruction structures (i) The region of the unpaired nucleotide includes at least a hairpin loop before or after it, (ii) comprising at least one target hybridization region comprising at least 2 nucleotides, preferably 3 nucleotides or more, single-stranded regions before or after a double-stranded region of at least 5 nucleotides, preferably 10 nucleotides or more, wherein at least one target hybridization region completely hybridizes with each of two or more hybridization regions of the artificial circular RNA, (d) When two or more hybridization regions in an artificial circular RNA hybridize with a target hybridization region, the hybridization energy measured by RNAcofold between the hybridization region and at least one target hybridization region is negative compared to the energy of the target disruption region, thereby disrupting the target disruption structure.

[0013] Preferably, the artificial circular RNA according to the first embodiment or any of its embodiments comprises 6 to 20 hybridization regions. Preferably, at least two, and preferably all, of the hybridization regions can completely hybridize with the same target hybridization region. Preferably, at least two, and preferably all, of the hybridization regions have different nucleotide sequences.

[0014] Preferably, in the artificial circular RNA according to the first embodiment or any of its embodiments, two or more hybridization regions are a) Separated by non-hybridization regions up to 20 nucleotides in size, or b) Not separated by non-hybridization regions, or c) It is duplicated.

[0015] Preferably, in the artificial circular RNA according to any one of the first aspect or its embodiments, one or more RNA fragments are selected from mRNA, tRNA, rRNA, non-coding RNA, and viral genomic RNA.

[0016] Preferably, in the artificial circular RNA according to any one of the first aspect or its embodiments, one or more RNA fragments are viral genomic RNA.

[0017] Preferably, in the artificial circular RNA according to any one of the first aspect or its embodiments, one or more RNA fragments are plus-strand single-stranded viral genomic RNA.

[0018] Preferably, in the artificial circular RNA according to any one of the first aspect or its embodiments, the viral genomic RNA is selected from influenza virus, HAV, poliovirus, coxsackievirus B, coronavirus, and rhinovirus (common cold).

[0019] Preferably, in the artificial circular RNA according to any one of the first aspect or its embodiments, the viral genomic RNA is selected from hepatitis C virus, dengue, Zika, chikungunya, West Nile, and yellow fever virus.

[0020] Preferably, in the artificial circular RNA according to any one of the first aspect or its embodiments, the viral genomic RNA is derived from severe acute respiratory syndrome coronavirus 2 (SARS-CoV- 2).

[0021] Preferably, in the artificial circular RNA according to any one of the first aspect or its embodiments, at least one or more target disruption structures are selected from the group consisting of: (a) Hepatitis C virus-derived internal ribosome entry (IRES) domain IV and domain V, capsid coding region hairpin element (cHP) or SL427, (b) Short stem loop (sHP) or capsid coding region hairpin element (cHP) derived from dengue virus, (c) 5’untranslated region (5’UTR), repetitive sequence element (RSE) or recoding element derived from chikungunya, (d) Stem loop III (SLIII) derived from West Nile, and / or (e) SL-2, replication site, target A, target C, target D derived from coronavirus, preferably from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).

[0022] Preferably, in the artificial circular RNA according to any of the first aspect or its embodiments, at least one or more target disrupting structures include target disrupting structures selected from the group consisting of SEQ ID NOs: 76, 77, 78 and / or 79 derived from hepatitis C virus, and the target hybridization regions are respectively selected from SEQ ID NOs: 25, 26, 27 and 28 for each of these target disrupting structures.

[0023] Preferably, in the artificial circular RNA according to any of the first aspect or its embodiments, at least one or more target disrupting structures include target disrupting structures selected from the group consisting of SEQ ID NOs: 29 and / or 30 derived from dengue virus, and the target hybridization regions are respectively selected from SEQ ID NOs: 29 and 30 for each of these target disrupting structures.

[0024] Preferably, in the artificial circular RNA according to any of the first aspect or its embodiments, at least one or more target disrupting structures include target disrupting structures selected from the group consisting of SEQ ID NOs: 80, 81 and / or 82 derived from chikungunya virus, and the target hybridization regions are respectively selected from SEQ ID NOs: 33, 35 and 31 for each of these target disrupting structures.

[0025] Preferably, in the artificial circular RNA according to the first embodiment or any of its embodiments, at least one target disruption structure includes or comprises the target disruption structure of Sequence ID No. 83 derived from the West Nile, and the target hybridization region is Sequence ID No. 37.

[0026] Preferably, in the artificial circular RNA according to the first embodiment or any of the embodiments thereof, at least one target disruption structure includes or comprises a target disruption structure selected from the group consisting of SEQ ID NOs. 84, 58, 85, 86 and / or 87 derived from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), and the target hybridization region is selected for each of these target disruption structures from SEQ ID NOs. 62, 58, 59, 60 and 61, respectively.

[0027] Preferably, in the artificial circular RNA according to the first embodiment or any of its embodiments, at least one target disruption structure is selected from the group consisting of SEQ ID NOs: 30 and / or 79 derived from dengue virus and hepatitis C virus, and the target hybridization region is selected from SEQ ID NOs: 30 and 28 for each of these target disruption structures, respectively.

[0028] Preferably, in the artificial circular RNA according to the first embodiment or any of its embodiments, At least one target disruption structure includes or comprises a target disruption structure selected from the group consisting of SEQ ID NOs: 30 and / or 83 derived from dengue virus and West Nile virus, and the target hybridization region is selected from SEQ ID NOs: 30 and 37 for each of these target disruption structures, respectively.

[0029] Preferably, the sequences of the artificial circular RNA according to the first embodiment or any of its embodiments are: SEQ ID NOs: 2, 3, 4, 5, 6 (for hepatitis C virus); SEQ ID NOs: 8, 9, 10 (for dengue virus); SEQ ID NOs: 12, 13, 14, 15, 39 (for chikungunya virus); SEQ ID NOs: 16 and 17 (broad-spectrum activity against both hepatitis C virus and dengue virus); SEQ ID NOs: 24 and 19 (for West Nile virus); SEQ ID NOs: 21, 22, and 23 (dengue virus) Broad-spectrum activity against both hepatitis C virus and West Nile virus); SEQ ID NO: 32 (broad-spectrum activity against both hepatitis C virus and dengue virus); SEQ ID NOs: 36, 38, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 65, 66, 67, 68, 69, 70, 71, 72 (in the case of severe acute respiratory syndrome coronavirus 2) comprises, or preferably consists of, the following nucleotides:

[0030] Preferably, one or more target hybridization regions of the artificial circular RNA that fully hybridize with two or more hybridization regions are included in the artificial RNA defined by SEQ ID NO: 1, SEQ ID NO: 7, SEQ ID NO: 11, SEQ ID NO: 34 and / or SEQ ID NO: 20.

[0031] In a second aspect, the present invention relates to a composition comprising the artificial RNA of the present invention.

[0032] In a third aspect, the present invention relates to a kit comprising the artificial RNA and / or composition of the present invention.

[0033] In a fourth aspect, the present invention relates to artificial RNA and / or compositions used as pharmaceuticals, preferably for use in methods for preventing and / or treating viral infections. Preferably, viral infections are caused by hepatitis C virus, hepatitis A virus, poliovirus, influenza virus, coxsackie B virus, rhinovirus (common cold), dengue fever, Zika, chikungunya, West Nile virus, yellow fever virus, or coronaviruses, such as SARS and / or MERS, preferably SARS-CoV-2.

[0034] In a sixth aspect, the present invention provides a method for screening artificial circular RNAs comprising two or more hybridization regions capable of disrupting one or more target disruption structures of one or more RNA fragments by hybridization, wherein the target disruption structures are i. A first region having at least a hairpin loop before or after the second region of an unpaired nucleotide; and ii. It is defined as including at least one target hybridization region containing at least 2 nucleotides, preferably 3 nucleotides or more, single-stranded regions before or after a double-stranded region of at least 5 nucleotides, preferably 10 nucleotides or more. This method, a) A step of identifying two or more hybridization regions of an artificial circular RNA as regions having a total length of 7 to 100 nucleotides, preferably 10 to 50 nucleotides, wherein when hybridizing with at least one target hybridization region, the hybridization energy between the two or more hybridization regions and at least one target hybridization region is negative compared to the energy of the target disruption structure, thereby disrupting one or more target disruption structures, and the two or more hybridization regions contained in the artificial circular RNA are NUPACK, RNAifold, or MoiRNAi The process involves identification using RNA reverse folding tools such as Fold. b) A step of designing an artificial circular RNA comprising two or more hybridization regions capable of disrupting one or more target disruption structures identified in step a), wherein the artificial circular RNA is 150 to 800 nucleotides long, preferably 200 to 600 nucleotides, and c) optionally includes the step of selecting artificial circular RNAs that can disrupt one or more target disruption structures designed in step b) by hybridization, and optionally packaging them in a product. [Brief explanation of the drawing]

[0035] [Figure 1] Figure 1 schematically illustrates the intracellular generation of antiviral circRNA. This figure describes how circular RNA can be generated within target cells. First, the figure shows a plasmid containing the CMV promoter, two complementary and repeating regions surrounding the candidate circular RNA, and adjacent splicing acceptor and donor sites. This plasmid is transfected into target cells. The cells then transcribe linear RNA as shown, and the region between the splicing donor and acceptor sites is circularized by reverse splicing.

[0036] [Figure 2] RNA synthesis and cyclization. A workflow consisting of four main steps: (A) Generation of a template adjacent to the T7 promoter by PCR. (B) RNA preparation by in vitro transcription. (C) Generation of specific ends for ligation. The in vitro transcribed RNA must be dephosphorylated before in vitro cyclization. (D) Ligation with T4 RNA ligase 1 or circRNA ligase. Circular or linear oligomers can be formed as byproducts.

[0037] [Figure 3]Figure 3 schematically illustrates the mechanism of action. This figure shows the mechanism of action of circular RNA that disrupts the viral cycle. Viruses contain RNA structures in their genomes that are essential for their life cycle. Circular RNA targets these structures by binding to them in a way that causes conformational changes that disrupt critical stages of the viral life cycle.

[0038] [Figure 4] Figure 4 schematically illustrates an example of a relevant operating mode. This figure illustrates how the hybridization of circular RNA can alter the viral cycle. Firstly, RNA-binding proteins (RBPs) are required at many stages of the viral life cycle. These RBPs generally bind to specific regions of the viral genome that require both sequence and structure (or structural condition). As shown in the figure, one example is the binding of a PTB to the last domain of an IRES element in many picornaviruses, which binds to the box of a single-stranded pyrimidine just 3' of the stable hairpin. As shown in the right figure, when the circular RNA binds to a specific structural region of the IRES and the single-stranded region preceding this structural region, the RNA structure is altered such that the pyrimidine box is no longer in the same electronic structure condition, and therefore the RBP cannot bind to it.

[0039] [Figure 5] Figure 5 shows an example of circRNAs that target three different regions of a viral genome (or three different viral genomes) with two hybridization sequences per target region. Regions of the same color indicate that they target the same viral region.

[0040] [Figure 6] Figure 6 schematically shows the RNA secondary structures of the start and end regions of the HCV genome. The highlighted areas are regions designed to be targeted by circular RNA.

[0041] [Figure 7]Figure 7 schematically shows the results of four designed RNAs against the HCV genome. The circRNA IDs correspond to the target regions shown in the previous figure. As can be seen, infection is reduced by up to 20% compared to the control.

[0042] [Figure 8] Figure 8 schematically shows the RNA secondary structure of the DENV genome. The highlighted areas are regions designed to be targeted by circular RNA.

[0043] [Figure 9] Figure 9 schematically shows the results of three designed RNAs on the DENV genome. The circRNA IDs correspond to the target regions shown in the previous figure. As can be seen, infection is reduced by up to 40% compared to the control.

[0044] [Figure 10] Figure 10 shows the results of four designed RNAs against the CHIKV genome. As can be seen, infection is reduced by up to 50% compared to the control.

[0045] [Figure 11] Figure 11 shows the results of broad-spectrum circRNAs designed for both DENV and HCV genomes when used to treat DENV infection. HCV_CDS2 is one of the circRNAs in Example 1 and is used here as a negative control. DENV1_chp is one of the circRNAs in Example 2 and is used here as a positive control.

[0046] [Figure 12] Figure 12 shows the results of broad-spectrum circRNAs designed for both HCV and the HCV genome when used to treat DENV infection. HCV_CDS2 is one of the circRNAs in Example 1 and is used here as a positive control. DENV1_chp is one of the circRNAs in Example 2 and is used here as a negative control.

[0047] [Figure 13] Figure 13 shows the results of a second broad-spectrum circRNA designed for both DENV and HCV genomes when used to treat DENV infection. HCV_CDS2 is one of the circRNAs in Example 1 and is used here as a negative control. DENV1_chp is one of the circRNAs in Example 2 and is used here as a positive control.

[0048] [Figure 14] Figure 14 shows the results of a second broad-spectrum circRNA designed for both HCV and the HCV genome when used to treat DENV infection. HCV_CDS2 is one of the circRNAs in Example 1 and is used here as a positive control. DENV1_chp is one of the circRNAs in Example 2 and is used here as a negative control.

[0049] [Figure 15] Figure 15 shows the results for two designed RNAs, circ wnv_sIII 1 and circ wnv_sIII 2, against the West Nile virus (WNV) genome. As can be seen, infection is reduced compared to the control.

[0050] [Figure 16] Figure 16 shows the results of three broad-spectrum circRNAs designed for both WNV and DENV (dchp_wslI_A, dchp_wslI_B, and dchp_wslI_C) when used to treat WNV infection. Positive and negative controls from Examples 2 and 5 were used. As can be seen, all circRNAs showed inhibition.

[0051] [Figure 17]Figure 17 shows the results of three broad-spectrum circRNAs designed for both WNV and DENV (dchp_wslI_A, dchp_wslI_B, and dchp_wslI_C) when used to treat DENV infection. Positive and negative controls from Examples 2 and 5 were used. As can be seen, all circRNAs showed inhibition.

[0052] [Figure 18] Figure 18 illustrates the ability of the engineered RNA against HCV to inhibit chronically infected cells. CircRNA inhibits infectivity in chronically HCV-infected cells. Huh7 / Scr cells were infected with HCV and transfected with circ_hcv_cds2 at 48 hpi. Luciferase levels were measured after 2 days. The effect on infectivity was determined by the change in luciferase expression levels.

[0053] [Figure 19] Figure 19 shows the results of the designed RNA targeting the region required for HCV RNA replication and its ability to inhibit HCV replication. Circ_hcv_cds2, which targets the region required for HCV RNA replication, inhibits HCV RNA replication. Circ_hcv_cds2 inhibited luciferase levels 48 hours after infection when HCV RNA was translated and replicated, but did not inhibit them 4 hours after infection when HCV RNA was translated alone. The effect on infectivity is determined by the change in luciferase expression levels. Statistical significance was calculated using a t-test (* indicates p-value < 0.05).

[0054] [Figure 20]Figure 20 shows the results of designed DENV RNAs targeting regions necessary for DENV RNA replication and their ability to inhibit HCV replication. Circ_dv_3utr and circ_dv_cHP_v1, designed to target structures within the DENV RNA genome that direct RNA replication, inhibit DENV RNA replication. Circ_dv_3utr and circ_dv_cHP_v1 inhibit luciferase expression levels at 48 hours when the RNA genome is translated and replicated, but not at 8 hours when translated alone. All results were obtained from at least three biological replicate experiments. Statistical significance was calculated using t-tests (* represents <0.05).

[0055] [Figure 21] Artificial circRNA structure and mode of action. 1) CircRNA contains several different hybridization (brown region H) and segregation sequences (light gray region S). The hybridization sequences target the viral RNA genome (dark gray region), and the segregation sequences allow for structural flexibility and physical separation between the hybridization sequences. Note that all H and S sequences are distinct from each other. 2) The hybridization regions are designed to target and disrupt specific viral RNA genome structures, resulting in reduced infectivity. Hybridization begins in a single-stranded region, externally, or in a hairpin loop or pseudoknot, and terminates within a helix, resulting in the disruption of the helix.

[0056] [Figure 22] WNV structure used for circWNV design. 5'-UTR (top) and 3'-UTR (bottom) from the WNV genome are shown. Red indicates the hybridization region used in circWNV design. UTR: untranslated region [Adapted from Fernandez-Sanles et al., 2017, Front.Microbiol.8, 1-16].

[0057] [Figure 23]Schematic CHIKV structure used in the design of circCHIKV. Predicted 5'-UTR (A), RSE (B), and recoding element (C) are shown. In A and B, all structures are targeted by circRNA in C, and hybridization regions are shown in red. UTR: untranslated region; RSE: repeating sequence element. [Reprinted from Kendra et al., 2018, Virology 339, 200-212; and from Khan et al., 2002, J.Gen.Virol.83, 3075-3084].

[0058] [Figure 24] The designed broad-spectrum circRNA impairs both DENV and HCV infectivity. Cells were transfected with circRNA dv_chp_v1, circRNA hcv_cds2, or a broad-spectrum circRNA (circ dchp_hcv_cds2_2) containing hybridization sequences derived from both circRNA dv_chp_v1 and circRNA hcv_cds2. The cells were then infected with either DENV or HCV containing a luciferase reporter gene, and infectivity was measured after 48 hours. All results were obtained from at least three biological replicates. The effect on infectivity was determined by the change in luciferase expression levels. Statistical significance was calculated using a t-test (* indicates p-value < 0.05).

[0059] [Figure 25] This document describes a protocol for obtaining circular RNA in vitro.

[0060] [Figure 26] The results for circular RNAs for DNV and WNV generated in vitro, as well as for circular RNAs for HCV, are shown.

[0061] [Figure 27] This shows the types of target destruction structures (inner lines) and target hybridization regions (outer lines).

[0062] [Figure 28] The relationship between the target fracture structure, the target hybridization region, and the hybridization region is illustrated to demonstrate the mechanism of fracture by hybridization.

[0063] [Figure 29] Comparison of the cutting efficiency of head ribozymes on different hammers.

[0064] [Figure 30] Gels with different magnesium conditions.

[0065] [Figure 31] A gel exhibiting various IVT times.

[0066] [Figure 32] A gel that exhibits RNAse R efficacy without gel purification.

[0067] [Figure 33] This shows the efficacy results of circRNAs designed against SAR-Cov2.

[0068] [Figure 34] This demonstrates the effectiveness of in vitro purified circRNA against SAR-CoV2. Detailed description of the invention

[0069] definition In describing this disclosure, the following terms are used and are defined as set forth below.

[0070] The forms "a," "an," and "the" refer to multiple objects unless otherwise indicated by the context.

[0071] The term "approximately" in relation to a given quantity or quantity implies a deviation of ±5 percent.

[0072] The terms "artificial circular RNA," "circular RNA," or "circRNA" are used herein to refer to non-coding RNAs that form a covalently closed continuous loop with their 3' and 5' ends joined to each other, and therefore they lack a 5' cap and consequently a polyadenylated tail. Therefore, they are exonuclease-mediated. It is resistant to degradation and debranching enzymes, and confers greater stability to them compared to other RNAs.

[0073] The terms "complementary" and "complementarity" are interchangeable and refer to the ability of polynucleotides to form base pairs with one another. Base pairs are typically formed by hydrogen bonds between nucleotide units in antiparallel polynucleotide chains or regions. Complementary polynucleotide chains or regions can base pair in the Watson-Crick manner (e.g., A and T, A and U, C and G). 100% (or total) complementarity refers to a situation where each nucleotide unit of one polynucleotide chain or region can hydrogen bond with each nucleotide unit of a second polynucleotide chain or region. Incomplete (or partial) complementarity refers to a situation where some, but not all, nucleotide units of two chains or regions can hydrogen bond with one another, and this can be expressed as a percentage.

[0074] The term "hybridization" is used to refer to a structure formed by two independent strands of RNA that form a double-stranded structure through base pairing from one strand to the other. These base pairs are thought to be GC, AU, and GU (A-adenine, C-cytosine, G-guanine, U-uracil). As in the case of complementarity, hybridization can be whole or partial.

[0075] The terms "transfection" or "transfect" are used to refer to the uptake of circular RNA by a cell. A cell is "transfected" when circular RNA is introduced inside the cell membrane.

[0076] Stem-loop intramolecular base pairing ("stem-loop") is a pattern that can occur in single-stranded DNA, or more commonly, in RNA. This structure is also known as a "hairpin" or "hairpin loop." It occurs when two regions of the same strand form a base pair to create a double helix ending in an unpaired loop, which is usually complementary in nucleotide sequence when read in opposite directions. The resulting structure is an important component of many RNA secondary structures. Helix base pairing does not have to be complete, and stretches of single-stranded nucleotides called "bulges" or "internal loops" may exist. As an important RNA secondary structure, it can direct RNA folding, protect the structural stability of messenger RNA (mRNA), provide recognition sites for RNA-binding proteins, and function as a substrate for enzymatic reactions.

[0077] Internal loops (also called interior loops) in RNA are found where double-stranded RNA separates due to the absence of Watson-Crick base pairing between nucleotides. Internal loops can be classified as either symmetric or asymmetric, and some asymmetric internal loops are also known as bulges. Internal loops differ from stem loops because they occur in the center of the stretch of double-stranded RNA.

[0078] An "outer loop" is a stretch of single-stranded nucleotides that separates a hairpin loop or multiloop.

[0079] The "multiloop" branches out from the double-stranded region into several hairpin loops.

[0080] In relation to the present invention, the “target disruption structure” is preferably a stem loop (or hairpin loop) before or after a sequence of unpaired nucleotides (outer loop, bulge, or inner loop or multiloop, see Figure 27).

[0081] In relation to the present invention, the "target hybridization region" precedes the double-stranded region. Alternatively, it is a region within the target disruption structure, which is composed of subsequent single-stranded regions. Therefore, the target hybridization region is a region that, upon hybridization with another RNA strand, causes disruption of the structure to which it belongs.

[0082] "Disruption by Hybridization": Given a target disruption structure, a target hybridization region, and an artificial RNA that completely hybridizes with the target hybridization region, disruption by hybridization occurs when the energy of hybridization is more favorable (more negative) than the energy of the target disruption structure. During such hybridization, the structure of the target disruption structure changes, at least completely, with respect to the overlap between the target disruption structure and the target hybridization region. Such energy calculations can be performed using RNAcofold(Lorenz, Ronny and Bernhart, Stephan H. and Honer zu Siederdissen, Christian and Tafer, Hakim and Flamm, Christoph and Stadler, Peter F. and Hofacker, Ivo L., ViennaRNA Package 2.0, Algorithms for Molecular Biology, 6:1 26, 2011, doi:10.1186 / 1748-7188-6-26; Reuter, JS, & Mathews, DH(2010).RNAstructure:software for RNA secondary structure prediction and analysis.BMC Bioinformatics.11,129;Mathews,DH,et al.,''Predicting oligonucleotide affinity to nucleic acid targets'',RNA,1999 5:1458-1469), RNAstructure (https: / / rna.urmc.rochester.edu / RNAstructure.html), Mfold (http: / / unafold.rna.albany.edu / ?q=mfold, M. Zuker, ``Mfold web server for nucleic acid folding and hybridization prediction'', Nucleic Acids Res. 31(13), 3406-3415, 2003), or Vienna package(http: / / rna.tbi.univie.ac.at / ;Lorenz,Ronny and Bernhart,Stephan H. and Honer zu Siederdissen,Christian and Tafer,Hakim and Flamm,Christoph and Stadler,Peter F. and Hofacker,Ivo It can be obtained in silico using well-established software such as L., ViennaRNA Package2.0, Algorithms for Molecular Biology, 6:1 26, 2011, doi:10.1186 / 1748-7188-6-26).

[0083] "Viral conserved structures" refer to viral genome RNA structures that are conserved among members of the same species, genus, or family. These structures are sometimes characterized in the literature and experimentally verified. In cases where such experimental proof is unavailable, software exists that can predict such structures in silico. For example, all viral genomes of the same family can be aligned using, for example, ClustalW, and then RNAz (RNAz 2.0: Improved noncoding RNA detection, Gruber AR, Findeiβ S, Washietl S, Hofacker IL, Stadler PF. Pac Symp Biocomput. 15:69-79, 2010; Nucleic Acids Res. 1994 Nov 11;22(22):4673-4680, doi:10.1093 / nar / 22.22.4673, PMCID:PMC308517, PMID:7984417; CLUSTAL W: Improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties, and weight matrix choice (JD Thompson, DG Higgins, and TJ Gibson). RNAz predicts thermodynamically stable structured regions that are more likely to be predicted by comparison with random sequences of the same length and sequence composition (z-score), and further evaluates the regions by supporting compensatory and consistent mutations in sequence alignment. Such conserved structures are thought to be so due to their functional relevance in the life cycle of the virus. In Example 14, we show how these viral conserved structures were found.

[0084] The term 5'UTR refers to the region upstream of the code area, immediately preceding the start codon.

[0085] The term "IRES" is defined as an internal ribosome entry site, an RNA element that enables translation initiation in a cap-independent manner as part of the larger process of protein synthesis. In eukaryotic translation, initiation typically occurs at the 5' end of the mRNA molecule because 5' cap recognition is required for the assembly of the initiation complex. While IRES elements are often located in the 5' UTR, they can also be found in other locations on the mRNA.

[0086] The term CDS refers to the coding region of messenger RNA or viral genome. This is the region that codes for the corresponding protein.

[0087] The term 3'UTR refers to the region of the viral genome immediately following the stop codon, downstream of the CDS.

[0088] The term "cHP" refers to "capsid-coding region hairpin elements," which are known regions of the flavivirus genome found within CDS.

[0089] The term "RSE" stands for "conserved repetitive sequence element" and is a known structure found in the 3'UTR of several alphavirus genomes.

[0090] The "SRVVLC," a structural region essential to the viral life cycle, is a structural region of the RNA viral genome that is indispensable for viral replication, capsid formation, and / or translation. In other words, if it is destroyed, the virus cannot perform its essential functions in its life cycle, and its infectivity decreases.

[0091] "Administering" artificial RNA to cells includes any means that can transport nucleic acids across the cell membrane, such as transduction, transfecting, electroporation, translocating, fusion, phagocytosis, shooting, or ballistic methods.

[0092] A “homologous region” refers to a region of a different virus strain / serotype that shares common structural and / or functional characteristics. Homologous structure does not necessarily imply sequence identity. Those skilled in the art can identify homologous regions of other virus strains / serotypes, such as those defined in this application, by conventional means.

[0093] The degree of identity between two sequences can be determined by conventional methods, for example, by standard sequence alignment algorithms known at the level of the art, such as BLASTn (Altschul SF et al. Basic local alignment search tool. J Mol Biol. 1990 Oct 5;215(3):403-10). It can be decided by doing so.

[0094] In the claims, the word “comprising” and its variations are not intended to exclude other technical features, additives, components, or processes. Furthermore, the term “comprising” may also encompass the term “consisting of.”

[0095] Description of the Invention Generally, RNA fragments such as mRNA, tRNA, rRNA, non-coding RNA, viral RNA genomes, and viral mRNA contain highly structured regions (regions with secondary structures) that are either essential for their function or, even if not essential, can play a more or less important role in the function of the RNA fragment. These highly structured regions contain hairpin loops or regions of unpaired nucleotides before or after at least a portion of the hairpin loops. When the structure (secondary structure, etc.) of these hairpin loops or a portion of the hairpin loops changes, conformational changes occur within the RNA fragment. This will result in changes in the functionality of the RNA fragment.

[0096] Biological RNA is single-stranded and often forms complex and intricate base-pairing interactions due to the increased ability to form hydrogen bonds derived from extra hydroxyl groups in the ribose sugar. The secondary structure of RNA consists of a single polynucleotide folded within the same molecule. RNA base pairing occurs when RNA folds between complementary regions. Both single-stranded and double-stranded regions are commonly found in RNA molecules. Antiparallel strands form a helical shape. The four basic elements in RNA secondary structure are helices, loops, bulges, and junctions. Stem-loops, or hairpin loops, are the most common elements of RNA secondary structure. Stem-loops are formed when an RNA strand folds back on itself to form a double helix structure called a stem, and unpaired nucleotides form a single-stranded region called a loop.

[0097] The secondary structure of RNA can be predicted by either computational or experimental methods. Computationally, the RNA secondary structure can be predicted from one or more nucleic acid sequences using tools publicly available to those skilled in the art, such as the Wien package, as described above.

[0098] As outlined above, RNA structures, such as RNA secondary structures, are crucial in many biological processes, including the translational regulation of messenger RNA, the replication of single-stranded RNA viruses, and the function of structural RNA and RNA / protein complexes. In fact, for many RNA molecules, the secondary structure is extremely important for the correct function of the RNA, and is often more important than the actual sequence.

[0099] Therefore, changes in the secondary structure of an RNA fragment inevitably lead to changes in the functionality of that RNA fragment.

[0100] The artificial RNAs of the present invention, preferably circRNAs, can alter the secondary structure of the RNA fragments they target. Therefore, it is possible to modulate the functionality of target RNA fragments using the artificial RNAs of the present invention. For example, within the viral genome, there are highly structured regions essential to the viral life cycle (SRVVLC). If these regions are altered (e.g., destroyed) in their conformation or secondary structure, the virus will be unable (and preferably unable) to perform its essential functions in its life cycle. Therefore, altering the conformation or secondary structure of these regions essential to the viral life cycle reduces the infectivity of the virus, and ideally, renders it completely inactive. The same principle can be applied to other RNA fragments, for example, tR. Changes in the conformation of NA molecules can lead to a decrease (or increase) in the translation efficiency of their specific tRNA. For example, changes in the secondary structure of an mRNA molecule can lead to a decrease (or increase) in the binding affinity of that mRNA molecule to a specific protein or ribosome, and thus affect its translation rate. Structural changes in IRES elements in both viruses and cells can affect translation initiation. Structural changes in the UTR of mRNA can obscure the miRNA binding site and thus affect the translational regulation of such mRNA. Structural changes in intron regions can affect splicing, etc.

[0101] In particular, the artificial RNA of the present invention, preferably circRNA, can hybridize with specific regions within a target RNA fragment. By doing so, the artificial RNA of the present invention, preferably circRNA, can disrupt these specific target regions within the target RNA fragment. These “specific regions within the target RNA fragment” are so-called “target disruption structures.”

[0102] The targeted disruption structure includes at least a hairpin loop before or after the unpaired nucleotide region, and also includes a so-called "target hybridization region." The target hybridization region is a region within the targeted disruption structure that includes a single-stranded region of at least two nucleotides before or after the double-stranded region.

[0103] The artificial RNA, preferably circRNA, of the present invention hybridizes with a target hybridization region via a so-called "hybridization region." In other words, the artificial RNA, preferably circRNA, of the present invention comprises at least one hybridization region, which is a region of the artificial RNA that can fully hybridize with one or more target hybridization regions contained in the target disruption region of the target RNA fragment.

[0104] Hybridization (or hybridization) is the phenomenon in which two RNA molecules anneal to each other via base pairing interactions. In the context of this invention, only standard base pairings are considered (CG, AU, and GU). "Complete hybridization" is understood to mean that all nucleotides in the hybridization region of the artificial RNA anneal to all nucleotides in the target hybridization region.

[0105] When a hybridization region completely hybridizes with a target hybridization region, the hybridization energy between the hybridization region and the target hybridization region is negative compared to the energy of the target disruption region. When this occurs, the target disruption structure is disrupted, i.e., the secondary structure of the target RNA fragment changes. Consequently, the functionality of the target RNA fragment also changes, i.e., the functionality of the RNA fragment is regulated by the artificial RNA of the present invention. This is reproducible, and therefore, given that the target hybridization region is known as described below, candidate hybridization regions that completely hybridize with the target hybridization region can be easily found by using RNA design tools such as NUPACK or RNAiFold.

[0106] In a first aspect, the present invention relates to an artificial RNA, preferably circRNA, suitable for disrupting one or more target disruption structures of one or more RNA fragments by hybridization.

[0107] By "disrupting one or more targeted disruption structures of one or more RNA fragments by hybridization," in relation to the present invention, one or more targeted disruption structures of one or more RNA fragments The secondary structure of the structure is understood to change when the hybridization region of the artificial RNA of the present invention completely hybridizes with the corresponding target hybridization region of the target disruption structure.

[0108] The artificial RNA of the present invention, preferably circRNA, comprises at least one hybridization region as described in the present invention and may be of any length, as long as it is suitable for hybridizing one or more target disruption structures of one or more RNA fragments. Preferably, the RNA of the present invention, preferably circRNA, comprises 100 to 1000 nucleotides, more preferably 150 to 800 nucleotides, and even more preferably 200 to 600 nucleotides.

[0109] As described above, the artificial RNA of the present invention is preferably circular RNA and includes at least one hybridization region. The at least one hybridization region is a region of artificial RNA (i.e., a sequence of nucleotides) that completely hybridizes with at least one target hybridization region contained in one or more target disruption structures of one or more RNA fragments. The number of nucleotides in the hybridization region is not limited as long as it can completely hybridize with at least one target hybridization region, but preferably it has the same length as the target hybridization region contained in one or more target disruption structures, thereby enabling disruption of one or more target disruption structures (i.e., alteration of the secondary structure).

[0110] In a preferred embodiment, at least one hybridization region has a total of 7 to 100 nucleotides, more preferably 10 to 50 nucleotides, and even more preferably 15 to 35 nucleotides, for example, 15 to 25 nucleotides.

[0111] As described above, the hybridization region completely hybridizes with at least one target hybridization region contained in one or more target disruption structures of one or more RNA fragments. The target disruption structure is a region of an RNA fragment that includes at least a portion of a hairpin loop preceded or followed by a region of unpaired nucleotides. The target disruption structure includes at least one target hybridization region. The target hybridization region is a region (i.e., a sequence of nucleotides) that includes a single-stranded region of at least two nucleotides before or after a double-stranded region. The length of the target hybridization region is not limited as long as it includes a single-stranded region of at least two nucleotides before or after a double-stranded region and can completely hybridize with at least one hybridization region of the artificial RNA. Preferably, the single-stranded region of the target hybridization region contains three or more nucleotides, e.g., 3, 4, 5, 10, 15 or more nucleotides. Preferably, the double-stranded region of the target hybridization region contains five or more nucleotides, e.g., 5, 7, 10, 15, 20, 25 or more nucleotides.

[0112] Importantly, the hybridization region of the artificial RNA of the present invention, which is preferably circular RNA, fully hybridizes with at least one target hybridization region contained in one or more target disruption structures of one or more RNA fragments. Thus, at least one hybridization region of the artificial RNA of the present invention has exactly the same number of nucleotides as the at least one target hybridization region it hybridizes with. Therefore, at least one hybridization region of the artificial RNA of the present invention has a first region of a fixed number of nucleotides that fully hybridizes with the single-stranded region of the target hybridization region, and a second region of a fixed number of nucleotides that fully hybridizes with the double-stranded region of the target hybridization region.

[0113] In other words, in the first step, the single-stranded region of the target hybridization region is The first step involves annealing with specific nucleotides in the hybridization region of the artificial RNA, which is preferably circular RNA. In the second step, the double-stranded region of the target hybridization region is disrupted either before or after the single-stranded region, creating a new interaction between one strand of the double-stranded region of the target hybridization region and the specific nucleotides in the hybridization region of the artificial RNA, which then anneal to each other. Disruption of the double-stranded region of the target hybridization region occurs when the hybridization energy between at least one hybridization region and at least one target hybridization region is negative (more favorable) than the energy of the target disruption region. The hybridization energy and the ability to disrupt one or more target disruption structures can be measured, for example, using RNAcofold, and the identification of potential candidate hybridization regions of the artificial RNA of the present invention, characterized in that the hybridization energy between at least one hybridization region and at least one target hybridization region is negative (more favorable) than the energy of the target disruption region, can be easily identified by RNA reverse folding tools such as NUPACK, RNAifold, or MoiRNAiFold, as shown in Example 4.

[0114] As a result, at least the double-stranded region of the target hybridization region is disrupted, and therefore the target disruption structure is also disrupted. Thus, the structure of the target disruption structure changes, at least completely, with respect to the overlap between the target disruption structure and the target hybridization region. Consequently, the secondary structure of the target RNA fragment changes, and therefore its functionality also changes.

[0115] Hybridization between the hybridization region of artificial RNA and the target hybridization region of the targeted disruption structure of the RNA fragment can be tracked in vitro or in vivo. Changes in the secondary structure of the RNA fragment can also be confirmed using well-known techniques in the art, such as SHAPE (Poulsen, Line Dahl et al. "SHAPE Selection (SHAPES) enriched for RNA structure signal in SHAPE sequencing-based probing data." RNA (New York, NY) vol.21,5(2015):1042-52.doi:10.1261 / rna.047068.114) or PARIS (Lu Z, Gong J, Zhang QC. PARIS: Psoralen Analysis of RNA Interactions and Structures with High Throughput and Resolution. Methods Mol Biol. 2018;1649:59-84.doi:10.1007 / 978-1-4939-7213-5_4).

[0116] As a result, the at least one hybridization region contained in the artificial RNA of the present invention is further characterized in that, when hybridizing with a target hybridization region, the hybridization energy between the at least one hybridization region and the at least one target hybridization region is negative compared to the energy of the target disruption region, thereby disrupting the target disruption structure. As described above, those skilled in the art can predict the hybridization energy between the at least one hybridization region and the at least one target hybridization region and the energy of the target disruption region. Tools available for this prediction include, for example, RNAcofold (Lorenz, Ronny and Bernhart, Stephan H. and Honer zu Siederdissen, Christian and Tafer, Hakim and Flamm, Christoph) as detailed above. and Stadler,Peter F.and Hofacker,Ivo L.,ViennaRNA Package2.0,Algorithms for Molecular Biology,6:1 26,2011,doi:10.1186 / 1 748-7188-6-26;Reuter,JS,&Mathews,DH(2010).RNAstructure:software for RNA secondary structure prediction and analysis.BMC Bioinformatics.11,129, Mathews,DHet Well-established software such as al., "Predicting oligonucleotide affinity to nucleic acid targets", RNA, 1999 5:1458-1469), RNAstructure (https: / / rna.urmc.rochester.edu / RNAstructure.html), Mfold (http: / / unafold.rna.albany.edu / ?q=mfold, M. Zuker, "Mfold web server for nucleic acid folding and hybridization prediction", Nucleic Acids Res. 31(13), 3406-3415, 2003), or Vienna package (http: / / rna.tbi.univie.ac.at / ).

[0117] Preferably, the hybridization energy between at least one hybridization region and at least one target hybridization region and the energy of the target disruption region are calculated using RNAcofold software, preferably with the settings described in Mathews, DH, et al., "Predicting oligonucleotide affinity to nucleic acid targets", RNA, 1999 5:1458-1469. However, it should be noted that such calculations are unnecessary when RNA reverse folding tools such as NUPACK, RNAifold, or MoiRNAiFold are used to identify potential candidate hybridization regions of the artificial RNA of the present invention, characterized in that the hybridization energy between at least one hybridization region and at least one target hybridization region is negative (more favorable) than the energy of the target disruption region.

[0118] For a given RNA fragment, a person skilled in the art can identify potential target disruption structures, since these are regions of RNA containing at least a portion of a hairpin loop before or after a region of unpaired nucleotides. Many RNA structure prediction software programs are available to those skilled in the art that predict the secondary structure of a given RNA sequence. For example, we refer to Mathews, DH, et al. "RNA secondary structure See prediction.Current protocols in nucleic acid chemistry vol.Chapter11(2007):Unit11.2.doi:10.1002 / 0471142700.nc1102s28.

[0119] Furthermore, since the target hybridization region is included in the target disruption structure and, as defined above, contains a single-stranded region of at least two nucleotides before or after the double-stranded region, a person skilled in the art can identify one or more target hybridization regions within the target disruption structure.

[0120] Once the target hybridization region is identified, a person skilled in the art can design one or more hybridization regions that fully hybridize with the target hybridization region, as shown above. Similarly, a person skilled in the art can design several software programs for designing RNA sequences that can fully hybridize with a given RNA sequence, e.g., NUPACK (http: / / www.nupack.org / design / new, BRWolfe, NJPorubsky, JNZadeh, RMDirks, and NAPierce), Constrained multistat e sequence design for nucleic acid reaction pathway engineering'',J Am Chem Soc,139:3134-3144,2017;BRWolfe and NAPierce,''Sequence design for a test tube of “interacting nucleic acid strands”, ACS Synth Biol, 4:1086 - 1100, 2015; J.N. Zadeh, B.R. Wolfe, and N.A. Pierce, “Nucleic acid sequence design via efficient ensemble defect optimization” J Comput Chem, 32:439 - 452, 2011; and R.M. Dirks, M. Lin, E. Winfree, and N.A. Pierce, “Paradigms for computational nucleic acid design” Nucl Acids Res, 32:1392 - 1403, 2004. (see also), or RNAiFold (https: / / bioinformatics.bc.edu / clotelab / RNAiFold / , Juan Antonio Garcia - Martin, Peter Clote, Ivan Dotu, “RNAiFold: A constraint programming algorithm for RNA inverse folding and molecular design, J Bioinform Comput Biol 11(2):1350001, 2013; and Garcia - Martin JA, Dotu I, Clote P., “RNAiFold 2.0 A web server and software to design custom and Rfam - based RNA molecules”, Nucleic Acids Research Web Server issue, 2015, doi:10.1093 / nar / gkv460, “RNAiFold 2.0 A web server This involves knowing about "and software to design custom and Rfam-based RNA molecules" (see also). Furthermore, it is possible to consider specific target hybridization regions and determine whether specific target disruption structures can be disrupted by hybridization. This can be achieved using several publicly available RNA reverse folding or RNA design tools such as NUPACK (http: / / www.nupack.org / design / new), RNAiFold (http: / / bioinformatics.bc.edu / clotelab / RNAiFold / ), or its recent extension MoiRNAiFold (https: / / moiraibiodesign.com / design / ).

[0121] For example, in RNAiFold (and MoiRNAiFold), given any specific target disruption structure such as the one shown in Figure 28 (AAUAGAGUCCUGCCCAUUGGCGGG) and its target hybridization region (GAGUCCUGCC), the following input files can be used: #RNAscdstr ((((((((((&....))))))))).......... #RNAseqcon NNNNNNNNNN&AAUAGAGUCCUGCCCAUUGGCGGG

[0122] Here, the input ((((((((((&....))))))))))......... corresponds to well-established dot-bracket notation (see, for example, RNAlib-2.4.18:RNA Structure Notations(univie.ac.at)), and the input NNNNNNN&AAUAGAGUCCUGCCCAUUGGCGGG indicates that all "N"s correspond to the above "(" and must hybridize with the corresponding ")" shown above (any nucleotide A, C, G, or U). In other words, RNAiFold (and MoiRNAiFold) targets high It should return a solution (or multiple solutions) for a potential hybridization region that can actually destroy the target destruction structure AAUAGUCCUGCCCAUUGGCGGG during complete hybridization to the hybridization region GAGUCCUGCC.

[0123] In this case, the sample set of 10 solutions returned by the RNA reverse folding tool was as follows: TIFF2026102653000001.tif52170 Firstly, the target disruption structure can be disrupted by hybridization. Secondly, using the hybridization regions (bold), circRNAs that can disrupt the target disruption structure by hybridization can be constructed (by separating these regions from random polynucleotide regions).

[0124] Therefore, if, given these input instructions, the software tool returns a solution (or multiple solutions), it means that the target disruption structure can indeed be disrupted by hybridization. From the set of solutions, follow the circRNA generation of the present invention: take any number of these solutions (hybridization regions) and separate them by random nucleotide regions. Example 14 shows additional input files of several target disruption structures used as examples throughout this application.

[0125] In relation to the present invention, a hybridization region is a region within an artificial circular RNA that hybridizes with a target hybridization region in the RNA fragment of interest, thereby disrupting the target disruption structure (see Figure 28 for a depiction of how all these concepts relate to each other). In preferred embodiments, the energy of hybridization and the ability to disrupt one or more target disruption structures can be calculated using RNA reverse folding tools such as NUPACK, RNAifold, or MoiRNAiFold.

[0126] As described above, in a much more preferred embodiment, the artificial RNA of the present invention is circular RNA. In this sense, the inventors have found that the use of artificial RNA-based therapies disclosed herein, preferably circular RNA, has fundamental advantages over other RNA molecules:

[0127] - Stability. On the other hand, circRNAs are extremely stable because they cannot be utilized by cellular exonucleases. This stability simplifies their use in therapy, in contrast to other current RNA-based therapies that require chemical modifications, which are expensive, unnatural, and may raise toxicity concerns.

[0128] - Resistance to the emergence and selection of escape variants. The designed artificial RNA, e.g., circRNA, preferably (i) a longer sequence in which hybridization to the target hybridization region is not affected by a single mutation, and (ii) a viral RN It contains multiple hybridization regions that can completely hybridize to multiple target hybridization regions within the A genome and prevent the emergence of resistant mutants through the selection of a single mutation.

[0129] - Ability to address multiple targets. All current effective therapies involve molecules that target specific viral proteins and can therefore only cure a single infection. By including hybridization regions in a designed artificial RNA, such as circRNA, that can hybridize with different target hybridization regions contained in the targeted disruption structures of different RNA fragments, such as different viral genomes, we can simultaneously target different RNA fragments, such as different viruses. This broad-spectrum therapy is highly valuable for (i) simplifying the treatment of co-infections, and (ii) treating acute infections that share geographical location and initial symptoms, such as those caused by dengue virus, Zika virus, and chikuginya virus, or those caused by SARS-CoV-2 and influenza. In acute infections, early treatment is crucial to control the disease and epidemic. Broad-spectrum therapy enables treatment even before a final diagnosis is achieved. Furthermore, it would be desirable to use such therapies as a preventive measure during an epidemic.

[0130] - Minimizing the risk of drug resistance. Furthermore, to further minimize the risk of drug resistance, all hybridization regions in engineered artificial RNAs targeting the same viral target disruption structure, such as circRNA (see, for example, Figure 21), may, advantageously, include GU pairing, unlike the artificial RNAs of the present invention, such as circRNA. In contrast to DNA, where complementarity is required, GU pairing is an effective hybridization pair in RNA.

[0131] In further embodiments, the artificial RNA of the present invention, which is preferably circular RNA, comprises at least one hybridization region, the at least one hybridization region can hybridize to a target hybridization region from different target disruption structures within the same RNA fragment, or from different RNA fragments.

[0132] In preferred embodiments, the artificial RNA of the present invention, which is preferably circular RNA, comprises two or more hybridization regions, preferably 6 to 20 hybridization regions. In further preferred embodiments, at least two, and preferably all, of the hybridization regions can completely hybridize with the same target hybridization region. Thus, the artificial RNA according to this preferred embodiment has a higher probability of disrupting the targeted disruption structure of the RNA fragment, and is therefore more effective in regulating the functionality of the RNA fragment, particularly the mutable RNA fragment (e.g., viruses and / or tumors). Thus, having multiple hybridization regions primarily affects the ability of viruses / tumors to avoid mutations.

[0133] In another, more preferred embodiment, the RNA of the present invention, which is preferably circular RNA, comprises at least two hybridization regions, and at least two, preferably all, of the hybridization regions have different nucleotide sequences, i.e., they are different from each other with respect to nucleotide sequences. Thus, in this preferred embodiment, at least two hybridization regions, preferably all of the hybridization regions of the artificial RNA, are different from each other and they all target (can hybridize) the same target hybridization region ("many-to-one" approach). In this way, the artificial RNA according to this preferred embodiment has a higher probability of disrupting the target disruption structure of the RNA fragment, and is subsequently more effective in modulating the functionality of the RNA fragment. Alternatively, at least two hybridization regions of the artificial RNA, preferably all of the hybridization regions, are different from each other. Redylation regions can target (hybridize) different target hybridization regions from different target disruption structures, even if they originate from the same RNA fragment or from different RNA fragments.

[0134] In a more preferred embodiment, two or more hybridization regions of the artificial RNA of the present invention are a) Separated by non-hybridization regions up to 20 nucleotides in size, or b) Not separated by non-hybridization regions, or c) It is duplicated.

[0135] Preferably, one or more RNA fragments are selected from mRNA, tRNA, rRNA, non-coding RNA, and viral genomic RNA. More preferably, the RNA fragments are viral genomic RNA. Even more preferably, one or more RNA fragments are positive sense single-stranded (ss) viral genomic RNA.

[0136] The viral RNA genome and viral mRNA contain highly structured regions ("targeted disruption structures") that include at least a portion of a hairpin loop preceded or followed by a region of unpaired nucleotides essential for its function. These highly structured regions are preferably structural regions essential to the viral life cycle (SRVVLC). When these regions are disrupted, the virus becomes unable (and preferably unable) to perform its essential life cycle functions. Therefore, disrupting these regions reduces the infectivity of the virus and, ideally, renders it completely inactive.

[0137] The inventors have designed an artificial RNA, preferably circular RNA, that includes at least one (and preferably two or more) hybridization regions that hybridize to and disrupt at least one target hybridization region (e.g., one, two, or more) within at least one target disruption structure (e.g., one, two, or more) present in the viral genomic RNA, thereby reducing or inhibiting viral infection. As described above, in a preferred embodiment, the artificial RNA of the present invention includes at least two (and preferably more) hybridization regions that are distinct from each other and can completely hybridize to and disrupt one target hybridization region within one target disruption structure present in the viral genomic RNA, thereby reducing or even inhibiting viral infection. In this way ("many-to-one" approach), the efficiency of disruption is improved.

[0138] In one embodiment, one or more target disruption structures are contained within an IRES element of the viral genome. Preferably, the one or more target disruption structures contained within the IRES element of the viral genome are structural regions essential to the viral life cycle (SRVVLC).

[0139] In another embodiment, one or more target disruption structures are located in the 5'UTR and / or 3'UTR of the viral genome. Preferably, one or more target disruption structures located in the 5'UTR and / or 3'UTR of the viral genome are structural regions essential to the viral life cycle (SRVVLC).

[0140] In further embodiments, one or more target disruption structures are included in the CDS of the viral genome. Preferably, the one or more target disruption structures included in the CDS of the viral genome are structural regions essential to the viral life cycle (SRVVLC).

[0141] In a further embodiment, at least two hybridization regions of the artificial RNA are It can completely hybridize with at least one target hybridization region contained in at least one disruption structure located in any combination of two positions in the IRES element, 5'UTR region, CDS region, and / or 3'UTR region of the viral genome.

[0142] In further embodiments, the artificial RNA of the present invention, which is preferably circular RNA, can disrupt one or more target disruption structures contained in at least two viral genomic RNAs by hybridization. Preferably, the artificial RNA of the present invention can disrupt one or more structural regions essential to the viral life cycle contained in at least two viral genomic RNAs by hybridization.

[0143] In preferred embodiments, the destruction of one or more structured regions essential to the life cycle of the virus reduces the activity of the virus, and more preferably, the destruction of one or more structured regions essential to the life cycle of the virus completely inactivates the virus.

[0144] The viral genome used as the target RNA fragment (viral genome RNA) is not limited. Any viral genome having a target disruption structure (i.e., a region containing at least a hairpin loop before or after a region of unpaired nucleotides) can be a suitable target RNA fragment for the artificial RNA of the present invention. Examples of viral genomes that can be target RNA fragments according to the present invention are as follows: hepatitis C virus (HCV), influenza virus, hepatitis A virus (HAV), poliovirus, coxsackie B virus, coronavirus, rhinovirus (common cold), dengue virus, Zika virus, chikungunya virus, West Nile virus, and yellow fever virus.

[0145] In another preferred embodiment, the target disruption structure that the artificial RNA of the present invention, preferably the artificial circular RNA of the present invention, can disrupt is a target disruption structure contained in the 5'UTR of a viral genome. In a preferred embodiment, the target disruption structure of the 5'UTR region of the DENV (dengue virus) genome is a cHP (capsid-coding hairpin region).

[0146] In another preferred embodiment, the target disruption structure that the artificial RNA of the present invention, preferably the artificial circular RNA of the present invention, can disrupt is a target disruption structure contained in the IRES element of a viral genome. In a preferred embodiment, the target disruption structure of the IRES element of the HCV (hepatitis C virus) viral genome is IRES1 and / or IRES2.

[0147] In another preferred embodiment, the target disruption structure that the artificial RNA of the present invention, preferably the artificial circular RNA of the present invention, can disrupt is a target disruption structure contained in the CDS of a viral genome. In a preferred embodiment, the target disruption structure of the CDS of a viral genome is CDS1 and / or CDS2. Preferably, the target disruption structure is selected from regions SL388, SL427, SL588 and / or SL750 of the HCV genome. In a preferred embodiment, the target disruption structure is region SL427 and / or region SL588 of the HCV genome (SL represents stem-loop).

[0148] In another preferred embodiment, the target disruption structure that the artificial RNA of the present invention, preferably the artificial circular RNA of the present invention, can disrupt is a target disruption structure contained in the 3'UTR of a viral genome. In a preferred embodiment, the target disruption structure of the 3'UTR region of the DENV genome is an sHP (short stem-loop or short hairpin region). In a further preferred embodiment, the target disruption structure of the 3'UTR region of the WNV (West Nile virus) genome is an SL_II.

[0149] In another preferred embodiment, in a circular RNA according to any of the prior embodiments, The target disruption structure is found in a combination of two or more of the following in the viral genome: IRES elements, 5'UTR structured regions, CDS structured regions, or 3'UTR structured regions.

[0150] In another preferred embodiment, the target disruption structure of the CHIKV (chikungunya virus) genome is the RSE region and / or recoding element (RE).

[0151] In a more preferred embodiment, one or more target hybridization regions that completely hybridize with at least one hybridization region of the artificial RNA according to the present invention, preferably the artificial circular RNA of the present invention, are included in SEQ ID NO: 1. In particular, the target hybridization region includes a nucleotide sequence defined in one or more of SEQ ID NOs: 25-28, or a nucleotide sequence having at least 70% identity with the nucleotide sequences defined in SEQ ID NOs: 25-28, preferably at least 80%, more preferably at least 90%, and even more preferably at least 95% identity with the nucleotide sequences defined in SEQ ID NOs: 25-28, or a homologous region in another HCV strain / serotype.

[0152] In a preferred embodiment, one or more target hybridization regions to which at least one hybridization region of the artificial RNA according to the present invention, preferably the artificial circular RNA of the present invention, completely hybridize include a nucleotide sequence defined by SEQ ID NOs. 25-27 (combination circ_hcv_ires1, circ HCV2, and circ_hcv_cds1, see Table 1 below), or a nucleotide sequence having at least 70% identity with the nucleotide sequence defined by SEQ ID NOs. 25-27, preferably at least 80%, more preferably at least 90%, and even more preferably at least 95% identity with the nucleotide sequence defined by SEQ ID NOs. 25-27, or a homologous region in another HCV strain / serotype.

[0153] The degree of identity between two sequences can be determined by conventional methods, for example, by standard sequence alignment algorithms known to the art, such as BLASTn (Altschul SF et al. Basic local alignment search tool. J Mol Biol. 1990 Oct 5;215(3):403-10).

[0154] A “homologous region” refers to a region of a different virus strain / serotype that shares common structural and / or functional characteristics. Homologous structure does not necessarily imply sequence identity. Those skilled in the art can identify homologous regions of other virus strains / serotypes, such as those defined in this application, by conventional means.

[0155] [Table 1]

[0156] Table 1. CircRNAs designed against HCV: Different circRNAs designed against HCV are classified in the table along with information on the target disruption structure that disrupts them by hybridization, the sequence of the target hybridization region that completely hybridizes with the hybridization region of the artificial RNA, the sequence of the entire circRNA, and the number of hybridization regions present in each candidate (each artificial circular RNA). IRES: Internal ribosome entry site; CDS: Coding sequence; cHP: Capsid hairpin.

[0157] In a more preferred embodiment, one or more target hybridization regions that completely hybridize with at least one hybridization region of the artificial RNA according to the present invention, preferably the artificial circular RNA of the present invention, are included in SEQ ID NO: 7. In particular, the target hybridization region includes a nucleotide sequence defined in one or more of SEQ ID NOs: 29-30, or a nucleotide sequence having at least 70% identity with the nucleotide sequences defined in SEQ ID NOs: 29-30, preferably at least 80%, more preferably at least 90%, and even more preferably at least 95% identity with the nucleotide sequences defined in SEQ ID NOs: 29-30, or a homologous region in another DENV strain / serotype.

[0158] [Table 2]

[0159] Table 2. CircRNAs designed against DENV: Different circRNAs designed against DENV are classified in the table along with information on the target disruption structure that disrupts them by hybridization, the sequence of the target hybridization region that completely hybridizes with the hybridization region of the artificial RNA, the sequence of the entire circRNA, and the number of hybridization regions present in each candidate (each artificial circular RNA). UTR: Untranslated region cHP: Capsid region Hairpin

[0160] In a more preferred embodiment, one or more target hybridization regions that completely hybridize at least one hybridization region of the artificial RNA according to the present invention, preferably the artificial circular RNA of the present invention, are included in one or more of SEQ ID NOs: 1 and SEQ ID NOs: 7. In particular, the target hybridization region includes a nucleotide sequence defined in one or more of SEQ ID NOs: 30 and SEQ ID NOs: 28, or a nucleotide sequence having at least 70% identity with the nucleotide sequences defined in SEQ ID NOs: 30 and SEQ ID NOs: 28, preferably at least 80%, more preferably at least 90%, and even more preferably at least 95% identity with the nucleotide sequences defined in SEQ ID NOs: 30 and SEQ ID NOs: 28, or a homologous region in another DENV strain / HCV strain / serotype.

[0161] [Table 3]

[0162] Table 3. Broad-spectrum DENV-HCV circRNA. Information on the circ_dv_cHP_v1-circ_hcv_cds2 of the two target disruption structures (DENV cHP and HCV CDS), the target hybridization region, the complete circRNA sequence, and the number of hybridization regions in each circRNA. cHP: capsid region, hairpin. CDS: coding sequence.

[0163] In a more preferred embodiment, one or more target hybridization regions that completely hybridize with at least one hybridization region of the artificial RNA according to the present invention, preferably the artificial circular RNA of the present invention, are included in SEQ ID NO: 11. In particular, the target hybridization region includes a nucleotide sequence defined in one or more of SEQ ID NOs: 31, 33, and 35, or a nucleotide sequence having at least 70% identity with the nucleotide sequences defined in SEQ ID NOs: 31, 33, and 35, preferably at least 80%, more preferably at least 90%, and even more preferably at least 95% identity with the nucleotide sequences defined in SEQ ID NOs: 31, 33, and 35, or a homologous region in another CHIKV strain / serotype.

[0164] [Table 4]

[0165] Table 4. CircRNAs designed for CHIKV: Different circRNAs designed for CHIKV are classified in the table along with information on the target disruption structure, the sequence of the target hybridization region, the sequence of the entire circRNA, and the number of hybridization regions present in each candidate (in each artificial circRNA). UTR: Untranslated region. CDS: Coding sequence.

[0166] In a more preferred embodiment, one or more target hybridization regions that completely hybridize with at least one hybridization region of the artificial RNA according to the present invention, preferably the artificial circular RNA of the present invention, are included in SEQ ID NO: 20. In particular, the target hybridization region includes the nucleotide sequence defined in SEQ ID NO: 37, or a nucleotide sequence having at least 70% identity with the nucleotide sequence defined in SEQ ID NO: 37, preferably at least 80%, more preferably at least 90%, and even more preferably at least 95% identity with the nucleotide sequence defined in SEQ ID NO: 37, or a homologous region in another WNV strain / serotype.

[0167] [Table 5]

[0168] Table 5. CircRNAs designed for WNV: Different circRNAs designed for WNV are classified in the table, along with information on the target disruption structure, the sequence of the target hybridization region, the sequence of the complete circRNA, and the number of hybridization regions present in each candidate (in each artificial circRNA). SLIII: Stem-loop III. UTR: Untranslated region.

[0169] In a more preferred embodiment, one or more target hybridization regions that completely hybridize at least one hybridization region of the artificial RNA according to the present invention, preferably the artificial circular RNA of the present invention, are included in SEQ ID NOs: 20 and 7. In particular, the target hybridization region includes a nucleotide sequence defined in one or more of SEQ ID NOs: 30 and 37, or a nucleotide sequence having at least 70% identity with the nucleotide sequences defined in SEQ ID NOs: 30 and 37, preferably at least 80%, more preferably at least 90%, and even more preferably at least 95% identity with the nucleotide sequences defined in SEQ ID NOs: 30 and 37, or a homologous region in another DENV / WNV strain / serotype.

[0170] [Table 6]

[0171] Table 6. CircRNAs designed for WNV and DENV: Different circRNAs designed for WNV and DENV are classified in the table, along with information on the target disruption structure, the sequence of the target hybridization region, the sequence of the complete circRNA, and the number of hybridization regions present in each candidate (in each artificial circRNA). SLI: Stem-loop I. cHP: Capsid region hairpin

[0172] In a more preferred embodiment, one or more target hybridization regions that completely hybridize at least one hybridization region of the artificial RNA according to the present invention, preferably the artificial circular RNA of the present invention, are included in one or more of SEQ ID NOs: 1, SEQ ID NOs: 7, SEQ ID NOs: 11 and / or SEQ ID NOs: 20. In particular, the target hybridization region includes a nucleotide sequence defined in one or more of SEQ ID NOs: 25-31, 33 and 35, 37, or a nucleotide sequence having at least 70% identity with the nucleotide sequences defined in SEQ ID NOs: 25-31, 33 and 35, 37, preferably at least 80% identity, more preferably at least 90% identity, and even more preferably at least 95% identity.

[0173] In another preferred embodiment, the target disruption structure within the HCV viral genome is found in the IRES and / or CDS regions and / or combinations thereof. More preferably, the target disruption structure of the HCV genome is selected from a list of target disruption structures that include nucleotide sequences having at least 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the nucleotide sequence defined in SEQ ID NOs. 76, 77, 78, or 79, or any combination thereof.

[0174] In another preferred embodiment, the target disruption structure within the dengue virus genome is found in the 3'UTR and / or cHP and / or a combination thereof. More preferably, the target disruption structure of the DENV genome is selected from a list of target disruption structures that include nucleotide sequences having at least 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the nucleotide sequence defined in SEQ ID NO: 29 or 30, or any combination thereof.

[0175] In another preferred embodiment, the target disruption structure within the Zika virus genome is found in the 5'UTR and / or RSE and / or a combination thereof.

[0176] In another preferred embodiment, the target disruption structure within the chikungunya virus genome is found in the 5'UTR and / or RSE and / or a combination thereof. More preferably, the target disruption structure of the CHIKV genome is selected from a list of target disruption structures that include nucleotide sequences having at least 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the nucleotide sequence defined in SEQ ID NO: 80, 81, or 82, or any combination thereof.

[0177] In another preferred embodiment, the target disruption structure within the West Nile viral genome is a stem-loop III (SL1II). More preferably, the target disruption structure of the WNV genome comprises or consists of a nucleotide sequence having at least 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the nucleotide sequence defined by SEQ ID NO: 83.

[0178] In another preferred embodiment, the hybridization region of the artificial RNA of the present invention, which is preferably circular RNA, targets two or more target disruption structures present in a single viral genome (i.e., it can completely hybridize with target hybridization regions present in a single viral genome).

[0179] In certain embodiments, the artificial RNA of the present invention, preferably the artificial circular RNA of the present invention, has broad-spectrum activity against RNA virus genomes, preferably HCV, dengue fever, Zika, chikungunya, West Nile, and yellow fever virus genomes. In the present invention, "broad-spectrum activity" with respect to the artificial RNA, preferably the circular RNA of the present invention, means that the artificial RNA is effective against a wide range of RNA viruses, preferably HCV, dengue fever, Zika, chikungunya, West Nile, and yellow fever.

[0180] In a more preferred embodiment, one or more target hybridization regions that completely hybridize with at least one hybridization region of the artificial RNA according to the present invention, preferably the artificial circular RNA of the present invention, are included in SEQ ID NO: 34. In particular, the target hybridization region includes a nucleotide sequence defined in one or more of SEQ ID NOs: 58-62, or a nucleotide sequence having at least 70% identity with the nucleotide sequences defined in SEQ ID NOs: 58-62, preferably at least 80%, more preferably at least 90%, and even more preferably at least 95% identity with the nucleotide sequences defined in SEQ ID NOs: 58-62, or a homologous region in another SARS-CoV-2 strain / serotype.

[0181] In another preferred embodiment, the targeted disruption structures within the SARS-CoV-2 viral genome are found at targets A, B, C, D, 3'UTR, 5'UTR, and / or combinations thereof. More preferably, the targeted disruption structures of the SARS-CoV-2 genome are selected from a list of targeted disruption structures that include nucleotide sequences having at least 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the nucleotide sequences defined in SEQ ID NOs. 84, 58, 85, 86, or 87, or any combination thereof.

[0182] [Table 7] TIFF2026102653000009.tif247170TIFF2026102653000010.tif206170

[0183] Table 7. CircRNAs designed for the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) isolate Wuhan-Hu-1 (complete genome: SEQ ID NO: 34): Different circRNAs designed for SARS-CoV-2 are classified in the table along with information on the target disruption structure, the sequence of the target hybridization region, the sequence of the complete circRNA, and the number of hybridization regions present in each candidate (in each artificial circRNA).

[0184] In a more preferred embodiment, one or more target hybridization regions that completely hybridize at least one hybridization region of the artificial RNA according to the present invention, preferably the artificial circular RNA of the present invention, are SEQ ID NOs: 1, 7, 11 and / or included in one or more of Sequence ID No. 20. In particular, the target hybridization region includes a nucleotide sequence defined in one or more of Sequence ID Nos. 25-31, 33, 35, 37 and 58-62, or a nucleotide sequence having at least 70% identity with the nucleotide sequences defined in Sequence ID Nos. 25-31, 33 and 35, 37, preferably at least 80% identity with the nucleotide sequences defined in Sequence ID Nos. 25-31, 33, 35, 37 and 58-62, more preferably at least 90% identity, and even more preferably at least 95% identity.

[0185] All possible combinations of targeted disruption structures and / or targeted hybridization regions derived from the same or different viruses disclosed above are included herein as specific embodiments for the design of circRNA sequences of the present invention. It should be noted that for each targeted disruption structure (IRES, CDS, etc.) disclosed throughout the present invention, different corresponding hybridization regions may be used and / or combined as described herein to design circRNA.

[0186] In a second aspect, the present invention provides a composition comprising the artificial RNA of the present invention, either alone or in combination, in any of the embodiments disclosed herein.

[0187] Preferably, the composition is a pharmaceutical composition, and preferably comprises the artificial RNA of the present invention in any of the embodiments and one or more pharmaceutically acceptable carriers.

[0188] When intended for clinical use, pharmaceutical compositions are prepared in a form suitable for the intended use. Generally, this involves preparing compositions that are essentially free of pyrogens and other impurities that may be harmful to humans or animals.

[0189] Colloidal dispersion systems, such as macromolecular complexes, nanocapsules, microspheres, beads, and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes, can be used as delivery vehicles for artificial RNA, such as cyclic RNA. Commercially available lipid emulsions suitable for delivering the nucleic acids of this disclosure to tissues include Intralipid, Liposyn, Liposyn II, Liposyn III, Nutrilipid, and other similar lipid emulsions. A colloidal system for use as a delivery vehicle in vivo is liposomes (i.e., artificial membrane vesicles). The preparation and use of such systems are well known in the art. Exemplary formulations are disclosed in U.S. Patent Nos. 5,981,505; 6,217,900; 6,383,512; 5,783,565; 7,202,227; 6,379,965; 6,127,170; 5,837,533; 6,747,014; and International Publication No. 03 / 093449. In particular, cationic liposomal formulations containing lipofectamine can be used for delivery. Lipofectamine can be formulated together with neutral copolymers or helper lipids. For example, see U.S. Patent No. 7,479,573, Dalby et al. (2004) Science Direct, Methods 33:95-103, and Hawley-Nelson et al. (1993) Focus 15:73-79.

[0190] To stabilize the delivery vehicle and enable uptake by target cells, it is generally desirable to use appropriate salts and buffers. Buffers are also used when introducing recombinant cells (e.g., those transfected ex vivo with artificial RNA such as circular RNA) into a patient. The aqueous compositions of this disclosure comprise an effective amount of the delivery vehicle dissolved or dispersed in a pharmaceutically acceptable carrier or aqueous medium.

[0191] The phrases "pharmaceutically acceptable" or "pharmacologically acceptable" refer to animals or humans. This refers to molecular entities and compositions that do not cause harmful allergic reactions or other adverse reactions when administered to humans. As used herein, "pharmaceutically acceptable carrier" includes solvents, buffers, solutions, dispersions, coatings, antimicrobial and antifungal agents, isotonic agents and absorption retarders, etc., that are acceptable for use in formulating pharmaceuticals suitable for administration to humans. The use of such carriers and agents for pharmaceutically active substances is well known in the art.

[0192] Any conventional medium or agent is intended for use in a therapeutic composition insofar as it is incompatible with the active ingredient of this disclosure. Auxiliary active ingredients may also be incorporated into the composition, provided they do not inactivate the nucleic acids of the composition.

[0193] Pharmaceutical forms suitable for injection or catheter delivery include, for example, sterile aqueous solutions or dispersions, and sterile powders for the immediate preparation of sterile injection solutions or dispersions. Generally, these preparations are sterile and fluid enough to be easily injectable. The preparations must be stable under manufacturing and storage conditions and must be protected from microbial contamination such as bacteria and fungi. Suitable solvents or dispersion media may include, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, liquid polyethylene glycol, etc.), suitable mixtures thereof, and vegetable oils. Adequate fluidity can be maintained, for example, by the use of coatings such as lecithin, maintaining the required particle size in the case of dispersions, and using surfactants. Prevention of microbial action can be achieved by various antimicrobial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, thimerosal, etc. In many cases, it is preferable to include isotonic agents, such as sugar or sodium chloride. Sustained absorption of injectable compositions can be achieved by using absorption retarders, such as aluminum monostearate and gelatin, in the composition.

[0194] Sterile injectable solutions can be prepared by incorporating appropriate amounts of active compounds in a solvent, along with any other components as needed (e.g., those listed above), followed by sterilization by filtration. Generally, dispersions are prepared by incorporating various sterilized active ingredients into a sterile vehicle containing a basic dispersion medium and other desired components, as listed above. For sterile powders for the preparation of sterile injectable solutions, preferred preparation methods include vacuum drying and freeze-drying techniques, which yield powders of the active ingredient(s) and any additional desired components from a pre-sterilized filtered solution.

[0195] The compositions of the present invention can generally be formulated in neutral or salt form. Examples of pharmaceutically acceptable salts include acid addition salts (formed with free amino acids of proteins) derived from inorganic acids (e.g., hydrochloric acid or phosphoric acid) or organic acids (e.g., acetic acid, oxalic acid, tartaric acid, mandelic acid, etc.). Salts formed with free carboxyl groups of proteins can also be derived from inorganic bases (e.g., sodium hydroxide, potassium hydroxide, ammonium hydroxide, calcium hydroxide, or ferric hydroxide) or organic bases (e.g., isopropylamine, trimethylamine, histidine, procaine, etc.).

[0196] When formulated, the solution is preferably administered in a therapeutically effective amount in a manner compatible with the administered formulation. The formulation can be easily administered in various dosage forms such as injection solutions and drug-release capsules. In the case of parenteral administration in aqueous solutions, for example, the solution is generally adequately buffered, and the liquid diluent is first made isotonic using, for example, sufficient saline or glucose. Such aqueous solutions can be used, for example, for intravenous, intramuscular, subcutaneous, and intraperitoneal administration. Preferably, as is known to those skilled in the art, and especially in light of this disclosure, sterile aqueous media are used. As an example, a single dose can be dissolved in 1 ml of isotonic NaCl solution and added to 1000 ml of subcutaneous injection solution, or injected into the proposed injection site (see, for example, Remington's Pharmaceutical Sciences, 15th Edition, pp. 1035-1038 and 1570-1580). Depending on the condition of the subject, some variation in dosage will inevitably occur. The person responsible for administration will, in any case, determine the appropriate dose for each individual subject. Furthermore, for administration to humans, the formulation must meet the sterility, pyrogenicity, and general safety and purity standards required by the FDA Office's Biologies standards.

[0197] The pharmaceutical compositions of the present invention can also be contained in syringes, implantable devices, etc., depending on the intended mode of delivery and use. Preferably, the compositions comprising artificial RNA, such as artificial circular RNA, prepared as described herein, are unit dosage forms, which means an amount of the composition suitable for a single dose in a pre-measured form or a pre-packaged form.

[0198] With regard to administration, at least one therapeutically effective cycle of treatment with artificial RNA, such as artificial circular RNA, may be administered to a subject for the treatment of viral infections caused by, for example, HCV, dengue virus, Zika virus, chikungunya virus, West Nile virus, yellow fever virus, or coronavirus, such as SARS and / or MERS, preferably SARS-CoV-2.

[0199] The “therapeutic effective dose or amount” of a composition containing artificial RNA, such as artificial circular RNA, is intended to be the amount that, when administered as described herein, results in a treated condition, such as an improved recovery from viral infection, cancer, or hereditary disorder, or other positive therapeutic response.

[0200] A composition comprising multiple therapeutically effective doses of the artificial RNA of the present invention, such as the artificial circular RNA of the present invention, and / or one or more other therapeutic agents is administered. The compositions of the present invention are typically, though not necessarily, administered by injection (subcutaneous, intravenous, intra-arterial, or intramuscular), by infusion, or topically. Further modes of administration, such as intraperitoneal, intrathecal, intratumoral, intralymphatic, intravascular, intralesional, and transdermal, are also contemplated. In some embodiments, the pharmaceutical composition comprising the artificial RNA of the present invention, such as the artificial circular RNA, is administered topically. The pharmaceutical composition comprising the artificial RNA of the present invention, such as the artificial circular RNA, and other agents may be administered using the same or different routes of administration according to any medically acceptable method known in the art.

[0201] Pharmaceutical compositions containing the artificial RNA of the present invention, such as artificial circular RNA, may also be administered prophylactically, for example, to prevent viral infections and / or cancer and / or hereditary disorders.

[0202] The pharmaceutical composition comprising the artificial RNA and / or other agents of the present invention, such as artificial circular RNA, is a sustained-release formulation or a formulation administered using a sustained-release device. Such devices are well known in the art and include, for example, small implantable pumps that can provide delivery over time in a continuous steady-state manner at various doses to achieve a sustained-release effect in non-sustained-release pharmaceutical compositions.

[0203] Those skilled in the art will understand the conditions under which the composition comprising the artificial RNA of the present invention can effectively treat and / or prevent. The actual dose administered will vary depending on the subject's age, weight and general condition, as well as the severity of the condition being treated, the judgment of the medical professional, and the conjugate administered.

[0204] The therapeutically effective dose can be determined by those skilled in the art and adjusted to the specific requirements of each particular case. Compositions containing the artificial RNA of the present invention, such as artificial circular RNA, can be administered alone or in combination with one or more other therapeutic agents. Specific administration schedules are known to those skilled in the art or can be determined experimentally using routine methods. Exemplary administration schedules include, but are not limited to, five times a day, four times a day, three times a day, twice a day, once a day, three times a week, twice a week, once a week, twice a month, once a month, and any combination thereof. Preferred compositions are those requiring administration once a day or less. It is a finished product.

[0205] A composition containing the artificial RNA of the present invention, such as artificial circular RNA, can be administered before, simultaneously with, or after other drugs. When provided simultaneously with other drugs, the artificial RNA of the present invention can be provided in the same or different compositions. Therefore, the artificial RNA and one or more other drugs can be presented to an individual by concurrent therapy.

[0206] "Concurrent therapy" refers to administration to a subject in which the therapeutic effect of a combination of substances is induced in the subject receiving treatment. For example, concurrent therapy can be achieved by administering, according to a specific dosing regimen, a dose of a pharmaceutical composition containing the artificial RNA according to the present invention, such as artificial circular RNA, and a dose of a pharmaceutical composition containing at least one other agent, which is a combination of both. Similarly, the artificial RNA and one or more other therapeutic agents of the present invention can be administered in at least one therapeutic dose. The administration of separate pharmaceutical compositions can be done simultaneously or at different times (i.e., sequentially, in any order, on the same day or on different days), insofar as the therapeutic effect of the combination of these substances is induced in the subject receiving treatment.

[0207] In a third aspect, the present invention provides, in any of its embodiments, a kit comprising the artificial RNA or composition of the present invention and instructions for using the artificial RNA or composition.

[0208] Therefore, any of the artificial RNAs and / or compositions described herein may be included in the kit. For example, a circular RNA, such as that disclosed herein, may be included in the kit. The kit may also include one or more transfection reagents to facilitate the delivery of the artificial RNA to cells. Such a kit may also include components that preserve polynucleotides or protect them from degradation. Such components may be RNAse-free or protected against RNAse.

[0209] Such kits generally include separate containers for each individual reagent or solution, provided they are appropriate. A kit may include one or more containers for holding artificial RNA and other drugs. Suitable containers for the composition include, for example, bottles, vials, syringes, and test tubes. Containers can be formed from a variety of materials, including glass or plastic. Containers may have a sterile access port (for example, a container may be a vial with a stopper that can be punctured by a subcutaneous needle).

[0210] The kit may further include a container containing a pharmaceutically acceptable buffer, such as phosphate-buffered saline, Ringer's solution, or dextrose solution. The kit may also include other materials useful to the end user, such as other pharmaceutically acceptable formulation solutions, including buffers, diluents, filters, needles, and syringes or other delivery devices. The delivery device may be pre-filled with the composition.

[0211] The kit may also include a package insert containing instructions on how to treat viral infections with the artificial RNA of the present invention. The package insert may be an unapproved draft insert or an insert approved by the U.S. Food and Drug Administration (FDA) or other regulatory body.

[0212] As described above, preferably, the artificial RNA of the present invention that may be included in the composition or kit of the present invention is, in any of its embodiments, SEQ ID NOs: 2, 3, 4, 5, 6 (in the case of HCV); SEQ ID NOs: 8, 9, 10 (in the case of dengue virus); SEQ ID NOs: 12, 13, 14, 15, This includes nucleotide sequences defined by 39 (for chikungunya virus); SEQ ID NOs: 16 and 17 (broad-spectrum activity against both HCV and dengue virus); SEQ ID NOs: 24 and 19 (for West Nile virus); SEQ ID NOs: 21, 22 and 23 (broad-spectrum activity against both dengue and West Nile virus); SEQ ID NOs: 32 (broad-spectrum activity against both HCV and dengue virus), and SEQ ID NOs: 36, 38, 40, 41, 42, 88, 89, 65, 43, 44, 45, 66, 67, 46, 47, 48, 49, 68, 69, 70, 50, 51, 52, 53, 71, 72, 54, 55, 56, and 57 (for SARS-CoV-2 virus). These are target hybridization regions that completely hybridize with the hybridization regions contained in the artificial RNA described in the following examples.

[0213] Preferably, in any embodiment, one or more target hybridization regions that completely hybridize with at least one hybridization region in the artificial RNA of the present invention, which may be included in the composition and / or kit of the present invention, are included in SEQ ID NO: 1, SEQ ID NO: 7, SEQ ID NO: 11, SEQ ID NO: 20 and / or SEQ ID NO: 34.

[0214] In a fourth aspect, the present invention provides, in any of its embodiments, an artificial RNA of the present invention, or a composition or kit of the present invention for use as a pharmaceutical. Preferably, in any of its embodiments, the present invention provides an artificial RNA of the present invention, or a composition or kit of the present invention for use in a method for preventing and / or treating a viral infection. Preferably, the artificial RNA of the present invention has broad activity against two or more RNA viruses.

[0215] In further embodiments, the present invention provides, in any of its embodiments, an artificial RNA of the present invention, or a composition or kit of the present invention for use in a method for preventing and / or treating cancer.

[0216] In further embodiments, the present invention provides, in any of its embodiments, an artificial RNA of the present invention, or a composition or kit of the present invention, for use in a method for preventing and / or treating a genetic disorder.

[0217] In relation to the present invention, a hereditary disorder refers to a health problem caused by one or more abnormalities in the genome. This can be caused by mutations in a single gene (monogeneic) or multiple genes (polygeneic) or chromosomal abnormalities. Examples of hereditary disorders include: familial hypercholesterolemia, polycystic kidney disease, neurofibromatosis type 1, hereditary spherocytosis, Marfan syndrome, Huntington's disease, sickle cell anemia, cystic fibrosis, Tay-Sachs disease, phenylketonuria, mucopolysaccharidosis, lysosomal acid lipase deficiency, glycogen storage disease, galactosemia, Duchenne muscular dystrophy, or hemophilia.

[0218] Accordingly, in a fourth aspect, the present invention provides the use of the artificial RNA of the present invention, or a composition or kit of the present invention, in any of its embodiments, for producing pharmaceuticals for preventing and / or treating viral infections and / or cancer and / or hereditary disorders.

[0219] In preferred embodiments, the viral infection is caused by HCV, dengue fever, Zika, chikungunya, West Nile, yellow fever virus, or coronavirus, such as SARS and / or MERS, preferably SARS-CoV-2.

[0220] As is well known in the art (see, for example, International Publication No. 2017 / 222911), any known method of nucleic acid delivery can be used to administer the artificial RNA (e.g., immunogenic or non-immunogenic) disclosed herein to a subject. For example, the artificial RNA can be administered to i It can be administered by transfection in vivo. Alternatively, the artificial RNA can be administered by transfection in vivo and subsequent transfer of cells transfected with the artificial RNA into the target cells.

[0221] Artificial RNAs, such as circular RNAs, can be delivered together with nucleic acid carriers (e.g., cationic carriers) or nanoparticles (e.g., lipid nanoparticles, polymer nanoparticles, nanoparticles containing polymer-peptide combinations, or electrostatic complexes). Artificial RNAs can be delivered to targets using recombinant viruses, exosomes, liposomes, or other lipid vesicles, or cells engineered to secrete artificial RNAs such as circular RNAs.

[0222] Artificial RNAs, such as circular RNAs, can also be conjugated to targeted ligands (e.g., small molecules, peptides, or proteins) for local delivery to specific target sites (e.g., cells, tissues, or organs). Artificial RNAs can even be ligated to internalization sequences, protein transduction domains, or cell-permeable peptides to facilitate cell entry.

[0223] In a fifth aspect, the present invention relates to a method for producing the artificial RNA of the present invention, preferably the artificial circular RNA of the present invention, wherein the method is a) A step of synthesizing the artificial RNA of the present invention, b) The step of screening the artificial RNAs of step a) for artificial RNAs that can disrupt one or more target disruption structures of one or more RNA fragments, preferably SRVVLC, and / or prevent (ideally make impossible) the virus from performing its essential life cycle functions, i.e., artificial RNAs that can cause conformational changes in target RNA fragments, for example, to cause conformational changes in viral RNA that interfere with the essential functions of the virus's life cycle.

[0224] circRNA production Artificial RNA, such as circRNA, is designed using in-house software tools. These tools allow for the consideration of several design constraints, such as the GC content range of the sequence. Once the artificial RNA, such as circRNA, is designed, it can be generated intracellularly or by in vitro transcription.

[0225] (1) Intracellular generation of artificial RNA, such as circRNA. Commercially obtain the corresponding artificial RNA sequence (circRNA sequence, etc.) for the DNA morphology and clone it into cells for later transformation using the following process.

[0226] First, the designed artificial RNA sequence is cloned into a plasmid (e.g., pcDNA3-CIRS7 plasmid) between two sequences that mediate splicing and circularization, SA (splicing acceptor) and SD (splicing donor) (the clone was generously provided by Dr. Thomas B. Hansen of Aarhus University (Denmark) (Patent application: International Publication No. 2014 / 082644). This plasmid contains a gene that confers resistance to the antibiotic ampicillin. Next, the induced plasmid is transformed into XL1-Blue competent cells (Sigma). The transformed cells are grown in LB medium containing ampicillin (LB-Amp) for selection. Then, one of the selected colonies is grown in LB-Amp liquid medium for 36 hours for amplification. After this time, the culture is centrifuged to obtain bacterial cells, and the plasmid is purified using the NucleoSpin® Plasmid Kit (Macherey-Nagel).

[0227] When these induced plasmids are introduced into human cells, the transcription promoter CMV is recognized by the cellular transcription mechanism, and the mRNA is expressed using the host transcription mechanism. To generate cRNA, the obtained RNA is circularized by a host splicing mechanism that produces the designed circRNA (see Figure 1).

[0228] In vitro generation of artificial circRNA. CircRNA generation can also be achieved in vitro by obtaining the corresponding linear RNA through chemosynthesis or in vitro transcription, and then circularizing it with RNA ligase (see Figure 2).

[0229] A more efficient approach is to generate circRNA in vitro from a template adjacent to the T7 promoter. This plasmid contains an autocleaving ribozyme that, after in vitro transcription, cleaves to produce ends that enable circularization and circRNA generation. For each candidate, the T7 promoter plasmid was linearized using HindIII (FastDigest) at 37°C for 30 minutes. The purified linearized molecules were transcribed in vitro and purified from polyacrylamide gel. Subsequently, the RNA was circularized using RtcB (NEB), and the circularized molecules were selected after RNAse R (Lucigen) treatment at 37°C for 30 minutes (see Figure 25).

[0230] More specifically, circRNA was generated from a T7-derived plasmid containing the target circRNA surrounded at both ends by ribozymes, which are RNA molecules with self-cleaving ability. The inventors used eggplant latent viroid (ELVd) for the 5' and coral for the 3' as hammerhead pairs. Lower cleavage efficiency was observed with the 5' hammerheads, so to determine which hammerhead pair was more efficient, the inventors tested several ribozymes, both natural and artificial. As seen in Figure 29, all hammerheads tested produced the desired cleaved RNA molecules, with the exception of hammerheads 3 and 7, and had different cleavage efficiencies, with hammerheads 5-6-9 being the best. After extracting the bands of the corresponding cleaved RNA molecules from the gel, the inventors saw similar amounts with the three hammerheads, but decided to use artificial ribozyme 3 (ART-RBZ3) because the hammerheads behaved as predicted in silico.

[0231] Next, the inventors tested whether they could increase the partially cleaved / fully cleaved ratio by using different concentrations of magnesium in the buffer solution, but they did not observe any difference (Figure 30).

[0232] Once the hammerhead was established, and in order to continue generating the desired circRNA, the inventors cloned the circRNA sequence into a plasmid and linearized 10 micrograms using Hind III (Fast Digest) according to the manufacturer's instructions. The linearized DNA was purified with phenol-chloroform and further transcribed in vitro. Furthermore, the inventors tested the optimal amount of linearized plasmid and reaction time for use as a template. The inventors confirmed that using either 10 micrograms or 1 microgram of linearized DNA yielded the same amount of cleaved RNA molecules using T7 RNA polymerase (Takara) incubated for 6 hours or 3 hours (as recommended by the manufacturer) (Figure 31). Therefore, the inventors decided to perform IVT using 1 microgram of template and a 3-hour reaction.

[0233] The IVT was stopped and run in a urea gel for 4 hours to excavate the desired band (complete cut), from which RNA was further isolated. This final step resulted in a low yield of recovered RNA and required sufficient time to generate enough linear molecules to continue the protocol. To improve this, the inventors purified the IVT by chloroform / isoamyl alcohol extraction instead of running the gel, which yielded better results in terms of recovery.

[0234] Next, the inventors proceeded with the circularization of 10 micrograms of purified linear RNA using RtcB (New England Biolabs) at 37°C for 1 hour. To eliminate acyclic RNA morphology, the obtained RNA was treated with RNAse R (Lucigen) and purified according to the manufacturer's instructions. To validate the RNAse R treatment, the inventors performed urea gel purification and selected the circRNA band. As previously mentioned, urea gel purification resulted in a significant loss of RNA material. Therefore, the inventors decided to avoid gel purification and increase the RNAse R conditions (Figure 32) and purify as previously done in protocols to obtain a larger amount of circRNA. This modification of the protocol (see Figure 25) is important for achieving a significant amount of circRNA at a reasonable cost and time. Finally, the presence of circRNA was confirmed using a TBE-urea gel with only 1% material.

[0235] Mode of operation Artificial RNAs such as circRNAs are designed to hybridize with selected regions (target hybridization regions) present in the targeted disruption structures of one or more RNA fragments, such as RNA viral genomes. These targeted disruption structures are related to the functionality of the RNA fragment, as described above. For example, if the RNA fragment is an RNA viral genome, the targeted disruption structures may be related to multiple functions of the viral RNA, such as translation, replication, localization, and / or encapsulation, as well as infectivity. These signals can be found in the 5' and 3' untranslated regions and coding sequence regions (CDS) of the viral RNA genome. In one embodiment, these targeted disruption structures may be located within IRES elements (internal ribosome entry sites), which are involved in sequestering host ribosomes to initiate translation of viral proteins into several viral RNA genomes.

[0236] The mechanism of action, in contrast to the state of the art, is not only hybridization between the hybridization region of the artificial RNA and the target hybridization region of the target disruption structure, but also the structural changes caused during this hybridization change the secondary structure of the RNA fragment and ultimately affect its function. For example, viral RNA genomes and viral mRNAs contain highly structured regions that are essential for their functions. Binding of a designed artificial RNA targeting a disruption structure in the viral RNA genome results in the unfolding of the structure (change in secondary structure), which in turn results in a decrease in infectivity or even inhibition (see, for example, FIG. 3).

[0237] For example, disruption of a target disruption structure in an RNA virus genome can prevent the accessibility of RNA-binding proteins (RBPs) to their target viral RNA sequences. It should be noted that this strategy is different from that aimed at hybridizing to the exact binding site of the RBP (see, for example, FIG. 4).

[0238] Sometimes, an RBP binds to a single-stranded RNA region that is required to be seen, for example, immediately 3' of a stem-loop, in a particular structural context (see, for example, the left side of FIG. 4). In the case of the present inventors, hybridization with different target hybridization regions may disrupt this stem-loop (which is part of the target disruption structure), and the binding site remains accessible, but the structural change invalidates RBP binding (see, for example, the right side of FIG. 4).

[0239] Structure The structure of designed artificial RNAs such as circRNA is very flexible. Generally, it preferably hybridizes with at least one of one or more RNA fragments in the viral genome, preferably at least one present in several target disruption structures, preferably at least one present in several target hybridization regions, preferably several hyb It includes a redissociation region. Preferably, some of these hybridization regions in artificial RNA preferably do not perform any function other than separating some of the hybridization regions, and are preferably separated by quasi-random sequences in the sense that they enable compliance with design constraints (low secondary structure, specific GC content, etc.). For example, FIG. 5 shows an example of an artificial RNA, which is a circRNA, targeting three different target disruption structures of a viral genome having two hybridization regions per one target disruption structure. Regions having the same color represent the fact that they target the same viral target disruption structure.

[0240] However, as described above, these preferably some hybridization regions of artificial RNAs, such as circRNAs, targeting the same target hybridization region within a specific target disruption structure are preferably designed to be different from each other. This is possible because RNA enables three different base pairing types: G-C, A-U, and G-U. This fact also involves that viral escape (by mutation) from artificial RNA is more difficult because it needs to avoid all different hybridization regions simultaneously. Note that the hybridization requirement is different from the antisense requirement or the complementarity requirement.

[0241] It is also possible to design the hybridization regions of a single artificial RNA in order to target different regions of different RNA fragments, such as different regions of different RNA viruses, to obtain a broad-spectrum artificial RNA.

[0242] In a sixth aspect, the present invention provides a method for screening an artificial circular RNA comprising two or more hybridization regions capable of disrupting one or more target disruption structures of one or more RNA fragments by hybridization, wherein the target disruption structure is i. a first region having at least a hairpin loop before or after a second region of unpaired nucleotides; and ii. It is defined as comprising at least one target hybridization region, which includes at least 2 nucleotides, preferably 3 nucleotides or more, single-stranded regions before or after a double-stranded region of at least 5 nucleotides, preferably 10 nucleotides or more. This method, a) A step of identifying two or more hybridization regions of an artificial circular RNA as regions having a total length of 7 to 100 nucleotides, preferably 10 to 50 nucleotides, wherein when hybridizing with at least one target hybridization region, the hybridization energy between the two or more hybridization regions and at least one target hybridization region is negative compared to the energy of the target disruption structure, thereby disrupting one or more target disruption structures, and each of the two or more hybridization regions contained in the artificial circular RNA is identified by an RNA reverse folding tool such as NUPACK, RNAifold, or MoiRNAiFold. b) A step of designing an artificial circular RNA comprising two or more hybridization regions capable of disrupting one or more target disruption structures identified in step a), wherein the artificial circular RNA is 150 to 800 nucleotides long, preferably 200 to 600 nucleotides. c) optionally includes the step of selecting artificial circular RNAs that can disrupt one or more target disruption structures designed in step b) by hybridization, and optionally packaging them in a product.

[0243] As shown above, in step a) of the method according to the sixth aspect or any embodiment thereof, the identification of two or more hybridization regions by step a) is performed using an RNA reverse folding tool such as NUPACK, RNAifold, or MoiRNAiFold. This method is performed, and preferably, a) A step of generating one or more nucleotide sequences of two or more hybridization regions that have the potential to completely hybridize with at least one target hybridization region contained in one or more target disruption structures of one or more RNA fragments by providing an RNA reverse folding tool with at least both a target disruption structure / sequence and a target hybridization region. b) The process includes obtaining as output only one or more nucleotide sequences of the hybridization regions, in the case of hybridizing with a target hybridization region, such that the hybridization energy between two or more hybridization regions and at least one target hybridization region is negative compared to the energy of the target disruption region, thereby disrupting the target disruption structure.

[0244] In a preferred embodiment of the method according to the sixth aspect or any of its embodiments, the method includes a pre-step step of identifying the structure of one or more RNA fragments, including the following, as a target disruption structure: a) A first region having at least a hairpin loop before or after the second region of an unpaired nucleotide; and b) At least one target hybridization region comprising at least 2 nucleotides, preferably 3 nucleotides or more, single-stranded regions before or after a double-stranded region of at least 5 nucleotides, preferably 10 nucleotides or more.

[0245] In a preferred embodiment of the method according to the sixth aspect or any embodiment thereof, the method includes a further step d) confirming in a biological setting the ability of the designed artificial circular RNA designed in step b) to disrupt one or more target disruption structures of one or more RNA fragments by hybridization.

[0246] In a preferred embodiment, the method according to the sixth aspect or any of its embodiments includes the use of a target destruction structure and a target hybridization region as defined above in Tables 1 to 7.

[0247] The present invention will be further described by reference to the following examples, which are not intended to limit the scope of the present invention. [Sequence Listing]

[0248] SEQ ID NO: 1 <211> 9678 <212> RNA <213>Hepatitis C virus <220> <221> misc_feature <222> 24..56 <223> / note= "Target region of 5'UTR" <220> <221> misc_feature <222> 323..346 <223> / note= "Target region of 5'UTR" <220> <221> misc_feature <222> 372..399 <223> / note= "Target region of CDS" <220> <221> misc_feature <222> 459..474 <223> / note= "Target region of CDS" <220> <221> misc_feature <222> 651..668 <223> / note= "Target region of CDS" <220> <221> misc_feature <222> 9567..9600 <223> / note= "Target region of 3'UTR" <400> 1 accugccccu aauaggggcg acacuccgcc augaaucacu ccccugugag gaacuacugu 60 cuucacgcag aaagcgccua gccauggcgu uaguaugagu gucguacagc cuccaggccc 120 cccccucccg ggagagccau aguggucugc ggaaccggug aguacaccgg aauugccggg 180 aagacugggu ccuuucuugg auaaacccac ucuaugcccg gccauuuggg cgugcccccg 240 caagacugcu agccgaguag cguuggguug cgaaaggccu ugugguacug ccugauaggg 300 cgcuugcgag ugccccggga ggucucguag accgugcacc augagcacaa auccuaaacc 360 ucaaagaaaa accaaaagaa acaccaaccg ucgcccagaa gacguuaagu ucccgggcgg 420 cggccagauc guuggcggag uauacuuguu gccgcgcagg ggccccaggu ugggugugcg 480 cacgacaagg aaaacuucgg agcgguccca gccacguggg agacgccagc ccauccccaa 540 agaucggcgc uccacuggca aggccugggg aaaaccaggu cgccccuggc cccuauaugg 600 gaugaggga cucggcuggg caggauggcu ccugucccccc cgaggcucuc gcccccuccug 660 ggggccccacu gacccccggc auaggucgcg caacgugggu aaagucaucg acacccuaac 720 gugggcuuu gccgaccuca ugggguacau ccccgucgua ggcgccccgc uuaguggcgc 780 cgccagagcu gucgcgcacg gcgugagagu ccuggaggac gggguuaauu augcaacagg 840 900 960 1020 cccgugcgag agaguggga auacgucacg gugugggug ccagucucgc caaacauggc 1080 uggggggcag cccggugcccc ucacgcagggg ucugcggacg caaucgaua uggugugau 1140 guccgccacc uucugcucug cucuacgu ggggggaccuc uggggcgggg ugaugcucgc 1200 ggcccaggug uucaucgucu cgccgcagua ccacugguu gugcaagaau gcaauugcuc 1260 1320 gcccacggcc accaugaucc uggcguacgu gaugcgcguc cccgagguca ucauagacau 1380 cguuagcggg gcucacuggg gcgucauguu cggcuuggcc uacuucucua ugcagggagc 1440 guggcgaag gucauguca uccuucugcu ggccgcuggg guggacgcgg gcaccaccac 1500 cguuggaggc gcguugcac guuccaccaa cgugauugcc ggcguguuca gccauggccc 1560 ucagcagaac auucagcuca uuaacaccaa cggcaguugg cacaucaacc guacugccuu 1620 gaauugcaau gacuccuuga acaccggcuu ucucgcggcc uuguucuaca ccaaccgcuu 1680 uaacucguca ggguguccag ggcgccuguc cgccgccgc aacaucgagg cuuuccggau 1740 agggugggc acccuacagu acgagguaa ugucaccaau ccagaggaua ugaggccgua 1800 cugcuggcac uaccccccaa agccguggg cguagucccc gcgaggucug uggugggcccc 1860 aguguacugu uucaccccca gcccgguagu aguggcacg accgacagac guggagugcc 1920 caccuacaca uggggagaga augagacaga ugucuuccua cugaacagca cccgaccgcc 1980 gcagggcuca ugguucggcu gcacguggau gaacuccacu gguuucacca agacuugugg 2040 2100 ggauuguuuuu aggaagcauc cugaugccac uuauauuaag ugugguucug ggcccuggcu 2160 cacaccaaag ugccuggucc acuacccuua cagacucugg cauuacccu gcacagucaa 2220 uuuuaccauc uucaagauaa gaauguaugu aggggggguu gagcacaggc ucacggccgc 2280 2340 uccucuguug cacucuacca cggaaugggc cauccugccc ugcaccuacu cagacuuacc 2400 cgcuuuguca acuggucuuc uccaccuuca ccagaacauc guggacguac aauacaugua 2460 uggccucuca ccugcuauca caaaauacgu cguucgaugg gagugggugg uacucuuauu 2520 ccugcucuua gcggacgcca gagucugcgc cugcuugugg augcucaucu uguugggcca 2580 ggccgaagca gcauuggaga aguuggucgu cuugcacgcu gcgagugcgg cuaacugcca 2640 uggccuccua uauuuugcca ucuucuucgu ggcagcuugg cacaucaggg gucggguggu 2700 ccccuugacc accuauugcc ucacuggccu auggcccuuc ugccuacugc ucauggcacu 2760 gccccggcag gcuuaugccu augacgcacc ugugcacgga cagauaggcg uggguuuguu 2820 gauauugauc acccucuuca cacucacccc gggguauaag acccuccucg gccagugucu 2880 guggugguug ugcuaucucc ugacccuggg ggaagccaug auucaggagu ggguaccacc 2940 caugcaggug cgcggcggcc gcgauggcau cgcgugggcc gucacuauau ucugcccggg 3000 ugugguguuu gacauuacca aauggcuuuu ggcguugcuu gggccugcuu accucuuaag 3060 ggccgcuuug acacaugugc cguacuucgu cagagcucac gcucugauaa ggguaugcgc 3120 uuuggugaag cagcucgcgg gggguaggua uguucaggug gcgcuauugg cccuuggcag 3180 guggacuggc accuacaucu augaccaccu cacaccuaug ucggacuggg ccgcuagcgg 3240 ccugcgcgac uuagcggucg ccguggaacc caucaucuuc aguccgaugg agaagaaggu 3300 caucgucugg ggagcggaga cggcugcaug uggggacauu cuacauggac uucccguguc 3360 cgcccgacuc ggccaggaga uccuccucgg cccagcugau ggcuacaccu ccaaggggug 3420 gaagcuccuu gcucccauca cugcuuaugc ccagcaaaca cgaggccucc ugggcgccau 3480 aguggugagu augacggggc gugacaggac agaacaggcc ggggaagucc aaauccuguc 3540 cacagucucu caguccuucc ucggaacaac caucucgggg guuuugugga cuguuuacca 3600 cggagcuggc aacaagacuc uagccggcuu acggggguccg guacacgcaga ugaacucgag 3660 ugcugagggg gacuugguag gcuggcccag ccccccuggg accaagucuu uggagccgug 3720 3780 gagacgcggg gacaagcggg gagcauugcu cuccccgaga cccauuucga ccuugaaggg 3840 guccucgggg gggccggugc ucugcccuag gggccacguc guugggcucu uccgagcagc 3900 uggugugcu cggggcgugg ccaaauccau cgauuuucauc cccguugaga cacggacgu 3960 uguuacaagg ucucccacuu ucagugacaa cagcacgcca ccggcugugc cccagaccua 4020 ucaggucggg uacuugcaug cuccaacugg caguggaaag agcaccaagg ucccugucgc 4080 guaugccgcc cagggguaca aaguacuagu gcuuaacccc ucgguagcug ccacccuggg 4140 guuuggggcg uaccuaucca aggcacaugg caucaauccc aacauuagga cuggagucag 4200 gaccgugaug accgggagg caucacgua cuccacaauu ggcaauuuc ucgccgaugg 4260 gggcugcgcu agcggcgccu augacaucau caauugcgau gaugccacg cuguggaugc 4320 uaccuccauu cucggcaucg gaacgguccu ugaucaagca gagacagccg gggucagacu 4380 aacugugcug gcuacggcca caccccccgg gugugaca accccauc ccgaauaga 4440 aggguaggc cucggggcggg agggugaggc cuckoo cuckoo cuckoo cuckoo 4500 cugcaucaag ggagggac accugauuuu cugcacuca aagaaaagu gugacgagcu 4560 cgcggcggcc cucggggca ugggcuugaa uacuauagg ggcuggacgu 4620 cuccauaaua ccagcucagg gagauguggu ggucgucgcc accgacgccc ucaugacggg 4680 guacacugga gacuuugacu ccgugaucga cugcaugua gcggucaccc aagcugucga 4740 cucagcug gaccccaccu cucacuauac cucacacucu cucacacaag acgcugucuc 4800 acgcagucag cgccgcgggc gcacagguag aggaagacag ggcacuuaua gguauguuuc 4860 cacuggugaa cgagccucag gaauguuuga caguguagug cuuugugagu gcuacgacgc 4920 aggggcugcg uggacgauc ucacaccagc ggagaccacc gucaggcuua gagcguauuu 4980 caacacgcccc ggcuacccg ugugucaaga ccaucuugaa uuuugggagg caguuuucac 5040 cggccucaca cacauagacg cccacuuccu cucccaaaca aagcaagcgg gggagaacuu 5100 cgcguaccua guagccuacc aagcuacggu gugcgccaga gccaaggccc cucccccguc 5160 cugggacgcc auguggagu gccuggcccg acucaagccu acgcuugcgg gccccacacc 5220 ucuccuguac cguuugggcc cuauuaccaa ugaggucacc cucacacacc cugggacgaa 5280 guacaucgcc acaugcaugc aagcugaccu ugaggucaug accagcacgu ggguccuagc 5340 uggaggaguc cuggcagccg ucgccgcaua uugccuggcg acuggaugcg uuuccaucau 5400 cggccgcuug cacgucaacc agcgagucgu cguugcgccg gauaaggagg uccuguauga 5460 ggcuuuugau gagaugagg aaugcgccuc uagggcggcu cucaucgaag aggggcagcg 5520 gauagccgag auguugaagu ccaagaucca aggcuugcug cagcaggccu cuaagcaggc 5580 ccaggacaua caacccgcua ugcaggcuuc auggcccaaa guggcaacaau uuugggccag 5640 acacaugugg aacuucauua gcggcaucca auaccucgca ggauugucaa cacugccagg 5700 gaacccgcg guggcuucca ugauggcauu cagugccgcc cucaccaguc cguugucgac 5760 caguaccacc auccuucuca acaucauggg aggcuggua gcgucccaga ucgcaccacc 5820 cgcggggggcc accggcuuug ucgucagugg ccuggugggg gcugccgugg gcagcauagg 5880 ccuggguaag gugcuggugg acauccuggc aggauauggu gcgggcauuu cgggggcccu 5940 cgucgcauuc aaguaucaugu cuggcgagaa gcccuuaug gaauguca ucaaucuacu 6000 gccugggauc cugucuccgg gagcccgu gguggggguc aucugcgcgg ccauucugcg 6060 ccgccacgug ggaccggggg agggcgcggu ccaauggaug aacaggcuua uugccuuugc 6120 6180 ugugacccaa cuacuuggcu cucuuacuau aaccagccua cucagaagac uccacaauug 6240 gauaacugag gacugcccca ucccaugcuc cggauccugg cuccgcgacg uggggacug 6300 gguuugcacc aucuugacag auucaaaaa uuggcugacc ucuaaauugu uccccaagcu 6360 gcccggccuc cccuucaucu cuugucaaaa gggguacaag ggugugugg ccggcacugg 6420 caucaugacc acgcgcugcc cuugcggcgc caacaucucu ggcaaugucc gccuggcuc 6480 uaugaggauc acaggggccua aaaccugcau gaacaccugg caggggaccu uuccuaucaa 6540 uugcuacacg gagggccagu gcgcgccgaa accccccacg aacucaaga ccgccaucug 6600 gaggggcg gccucggagu acgcggaggu gacgcagcau gggucguacu ccuaguaac 6660 aggacugacc acugacauc ugaaaauucc ugccaacua ccuucuccag aguuuuucc 6720 cuggguggac gguguggaga uccauagguu ugcaccaca ccaagccgu uuuuccggga 6780 ugaggucucg ucugcguug ggcuuaauuc cuugcuguc gggucccagc ucccuguga 6840 accugagccc gacgcagacg uauugagguc caugcuaca gauccgcccc acaucacggc 6900 ggagacugcg gcgcggcgcu uggcacgggg aucaccucca ucugaggcga gcuccucagu 6960 gagccagcua ucagcaccgu cgcugcgggc caccugcacc accaccuauga acaccuauga 7020 cguggacaug gucgaugcca accugcucau ggggcggu guggcucaga cagagccuga 7080 guccagggug cccguucugg accuucucga gccaauggcc gaggagaga gcgaccuuga 7140 gcccucaaua ccaucggagu gcaugcuccc caggagcggg uuccacggg ccuuaccggc 7200 uugggcacgg ccugacuaca acccgccgcu cguggaaucg uggaggaggc cagauuacca 7260 accgcccacc gugcgugu gugcuccccc cccccccaag aaggccccga cgccuccccc 7320 aaggacgc cggacagugg gucugagcga gagcaccaua ucagaagccc uccagcaacu 7380 ggccaucaag accuuuggcc agccccccuc gagcggugau gcaggcucgu ccacgggggc 7440 gggcgccgcc gaauccggcg guccgacguc cccuggugag ccggcccccu cagagacagg 7500 uuccgccuccc ucuaugcccc cccucgaggg ggagccugga gauccggacc uggagucuga 7560 ucagguagag cuucaaccuc ccccccaggg gggggggggua gcuccccgguu cggggcucggg 7620 7680 cuggaccggg gcucuaauaa cucccuguag ccccgaagag gaaaguugc caaucaaccc 7740 uuugaguaac ucgcuguugc gauaccauaa caagguugaac ugaacaacau caaagagcgc 7800 cucacagagg gcuaaaaagg uaacuuuga caggacgcaa gugcucgacg cccauuauga 7860 cucagucuua aggacauuca agcuagcggc uuccaagguc agcgcaaggc uccucaccuu 7920 gggaggggcg ugccaguuga cuccacccca uucugcaaga uccaaguaug gauucggggc 7980 caaggagguc cgcagcuugu ccgggaggc cguuaaccac aucaaguccc ugggaaagga 8040 ccuccuggaa gacccacaaa caccaauucc cacaaccauc auggccaaaa augagguguu 8100 cugcguggac cccgccaagg gggguaagaa accagcucgc cucaucguuu acccugaccu 8160 cggcguccgg gucugcgaga aaauggcccu cuaugacauu acacaaaagc uuccucaggc 8220 gguaauggga gcuuccuaug gcuuccagua cuccccugcc caacgggugg aguaucucuu 8280 gaaagcaugg gcgggaaaaga aggaccccau ggguuuuucg uaugauaccc gaugcuucga 8340 8400 gcccgaggag gcccgcacug ccauacacuc gcugacugag agacuuuacg uaggagggcc 8460 cauguucaac agcaaggguc aaaccugcgg uuacagacgu ugccgcgcca gcggggugcu 8520 aaccacuagc auggguaaca ccaucacaug cuaugugaaa gcccuagcgg ccugcaaggc 8580 ugcggggaua guugcgccca caaugcuggu augcggcgau gaccuaguag ucaucucaga 8640 aagccagggg acugaggagg acgagcggaa ccugagagcc uucacggagg ccaugaccag 8700 guacucugcc ccuccuggug aucccccccag accggaauau gaccuggagc uaauaacauc 8760 cuguuccuca aaugugucug uggcguuggg cccgcggggc cgccgcagau acuaccugac 8820 cagagaccca accacuccac ucgcccgggc ugccugggaa acaguuagac acucccuau 8880 caauucaugg cugggaaaca ucauccagua ugcuccaacc auauggguuc gcaugguccu 8940 aaugacacac uucuucucca uucucauggu ccaagacacc cuggaccaga accucaacuu 9000 ugagauguau ggaucaguau acuccguga uccuuuggac uaauugagag 9120. snowflake 9120. snowflake 9120. snowflake ggcucagcc cucagaaac uuggggcgcc accccucagg guguggaga gucgggcucg cgcagucagg gcgucccuca ucucccgugg agggaagcg gccguuugcg gccgauaucu 9300. snow gcggugaaga snow snow snow snow snow snow snow snow snow snow ggacuuaucc cover ccgucggcgc cggcgggggc gacauuuuuc acagcguguc gcgcgcccga ccccgcucau uacucuucgg ccuacuccua cuuuucguag ggguaggccu snowflake cccgcucggu agagcggcac snowflake 9480. snowflake cccgcucggu 9540. 9540. 9540. 9540. 9540. 9540. 9540. 9540 9600. snow snow snow snow snow snow snow agucacggcu agcugugaaa gguccgugag ccgcaugacu gcagagagug ccguaacugg 9660 ucucucugca gaucaugu 9678 Sequence ID 2 <211> 294 <212> RNA <213> Artificial arrangement <220> <223> Circular artificial sequence for IRES1 (Hepatitis C virus) (circ_hcv_ires1) <220> <221> misc_feature <222> 1..33 <223> / note="Hybridization site for IRES (Hepatitis C virus)" <220> <221> misc_feature <222> 1..33 <223> / note="Hybridization site for IRES (Hepatitis C virus)" <220> <221> misc_feature <222> 43..75 <223> / note="Hybridization site for IRES (Hepatitis C virus)" <220> <221> misc_feature <222> 85..117 <223> / note="Hybridization site for IRES (Hepatitis C virus)" <220> <221> misc_feature <222> 127..159 <223> / note="Hybridization site for IRES (Hepatitis C virus)" <220> <221> misc_feature <222> 169..201 <223> / note="Hybridization site for IRES (Hepatitis C virus)" <220> <221> misc_feature <222> 169..201 <223> / note="Hybridization site for IRES (Hepatitis C virus)" <220> <221> misc_feature <222> 211..243 <223> / note="Hybridization site for IRES (Hepatitis C virus)" <220> <221> misc_feature <222> 253..285 <223> / note="Hybridization site for IRES (Hepatitis C virus)" <400> 2 uaguuccuca cgggggagu guucguggug gagcggcgcc aaugguuuuu ugcgggggag 60 ugguucgugg cggggguaac cccuuaguuc uucauggggg ggugguucau gguggggaaa 120 cuaccguagu uuuucacagg ggggugguuc auggcggaga gaagcgcuua guuucuuaug 180 ggggagugau ucauggcgga guguggggca ugguuuucg caggggagug auucguggug 240 gggagaguuu ugugguucuu uguagggggg ugauucgugg uggggacauc auug 294 Sequence ID 3 <211> 373 <212> RNA <213> Artificial arrangement <220> <223> Circular artificial sequence for IRES2 (Hepatitis C virus) <220> <221> misc_feature <222> 11..34 <223> / note="Hybridization site for IRES (Hepatitis C virus)" <220> <221> misc_feature <222> 44..67 <223> / note="Hybridization site for IRES (Hepatitis C virus)" <220> <221> misc_feature <222> 77..100 <223> / note="Hybridization site for IRES (Hepatitis C virus)" <220> <221> misc_feature <222> 110..123 <223> / note="Hybridization site for IRES (Hepatitis C virus)" <220> <221> misc_feature <222> 133..166 <223> / note="Hybridization site for IRES (Hepatitis C virus)" <220> <221> misc_feature <222> 176..199 <223> / note="Hybridization site for IRES (Hepatitis C virus)" <220> <221> misc_feature <222> 209..232 <223> / note="Hybridization site for IRES (Hepatitis C virus)" <220> <221> misc_feature <222> 242..265 <223> / note="Hybridization site for IRES (Hepatitis C virus)" <220> <221> misc_feature <222> 275..298 <223> / note="Hybridization site for IRES (Hepatitis C virus)" <220> <221> misc_feature <222> 308..331 <223> / note="Hybridization site for IRES (Hepatitis C virus)" <220> <221> misc_feature <222> 341..373 <223> / note="Hybridization site for IRES (Hepatitis C virus)" <400> 3 ugccgaaaau gcuuauggug cacgguuugc gagagcaaga agagcuugug gugcaugguu 60 ugcggggaca guaaaggcuc auggugcaug gucuacgagg ugagauagcg uuuauggugu 120 acggucugug ggaaauuccg guguucgugg ugugcggucu gugggauauu agaucguuca 180 ugguguacgg uuugugggga uaauagcggc uuauggugca cggucuaugg gacgcgagaa 240 ggcucauggu gugugguuug ugaggcagug ugcgguucau ggugcacggu cugcgagaca 300 ccaggcggcu cguggugcac ggucuauggg gugucgucgu gcuuguggug ugcggucuau 360 gagggcggcu gaa 373 Sequence ID 4 <211> 346 <212> RNA <213> Artificial arrangement <220> <223> Circular artificial sequence for CDS1 (Hepatitis C virus) (circ_hcv_cds1) <220> <221> misc_feature <222> 11..38 <223> / note="Hybridization site for CDS1 (hepatitis C virus)" <220> <221> misc_feature <222> 53..80 <223> / note="Hybridization site for CDS1 (hepatitis C virus)" <220> <221> misc_feature <222> 95..122 <223> / note="Hybridization site for CDS1 (hepatitis C virus)" <220> <221> misc_feature <222> 137..164 <223> / note="Hybridization site for CDS1 (hepatitis C virus)" <220> <221> misc_feature <222> 179..206 <223> / note="Hybridization site for CDS1 (hepatitis C virus)" <220> <221> misc_feature <222> 221..248 <223> / note="Hybridize against CDS1 (hepatitis C virus)" "Parts" <220> <221> misc_feature <222> 263..290 <223> / note="Hybridization site for CDS1 (hepatitis C virus)" <220> <221> misc_feature <222> 305..332 <223> / note="Hybridization site for CDS1 (hepatitis C virus)" <400> 4 cugcacuggg uuugggcgac gguugguguu uuuuuuggac gagauuucuu gcucugggcg 60 acgguuggug uuucuuuugg guguuagugc guacucuggg cgaugguugg uguuucuuuu 120 gguaauuauu acuuucuuug ggcgaugguu gguguuuuuu uugguggggu gaaaagaguu 180 ugggcggugg uugguguuuc uuuuggcgug gugaguaaca uuuggguggc gguugguguu 240 uuuuuuggcu gcaacgcacc gguuugggcg acgguuggug uuucuuuugg gggucaaccg 300 ggaaucuggg uggcgguugg uguuuuuuuu ggugauggcc gaggua 346 Sequence ID 5 <211> 355 <212> RNA <213> Artificial arrangement <220> <223> Circular artificial sequence (circ_hcv_combo1) for IRES1, IRES2, and CDS1 (hepatitis C virus) <220> <221> misc_feature <222> 11..43 <223> / note="Hybridization site for IRES1 (Hepatitis C virus)" <220> <221> misc_feature <222> 54..77 <223> / note="Hybridization site for IRES2 (Hepatitis C virus)" <220> <221> misc_feature <222> 88..115 <223> / note="Hybridization site for CDS1 (hepatitis C virus)" <220> <221> misc_feature <222> 126..158 <223> / note="Hybridization site for IRES1 (Hepatitis C virus)" <220> <221> misc_feature <222> 169..192 <223> / note="Hybridization site for IRES2 (Hepatitis C virus)" <220> <221> misc_feature <222> 203..230 <223> / note="Hybridization site for CDS1 (hepatitis C virus)" <220> <221> misc_feature <222> 241..273 <223> / note="Hybridization site for IRES1 (Hepatitis C virus)" <220> <221> misc_feature <222> 284..307 <223> / note="Hybridization site for IRES2 (Hepatitis C virus)" <220> <221> misc_feature <222> 318..345 <223> / note="Hybridization site for IRES2 (Hepatitis C virus)" <400> 5 gcgccaagua uaguuccuca caggggagug auuuauggug gagaccccua aacgcuugug 60 gugcacgguc uacgggguac cgagaaguuu gggcgguggu ugguguuuuu uuuggcgcuu 120 gugggugguu uuuugcaggg gagugauuua ugguggaggc aagaguuugc uuauggugua 180 cggucuguga gaugacauca gguuugggcg gcgguuggug uuuuuuuugg augaggaacc 240 uaguucuuca uaggggagug auuuguggug gagacagcga guuguuugug gugcacgguu 300 uacgagaaga augaagaucu ggguggcggu ugguguuucu uuugguagca caugu 355 Sequence ID 6 <211> 288 <212> RNA <213> Artificial arrangement <220> <223> Circular artificial sequence for CDS2 (Hepatitis C virus) (circ_hcv_cds2) <220> <221> misc_feature <222> 4..14 <223> / note="Hybridization site for CDS2 (Hepatitis C virus)" <220> <221> misc_feature <222> 28..38 <223> / note="Hybridization site for CDS2 (Hepatitis C virus)" <220> <221> misc_feature <222> 52..62 <223> / note="Hybridization site for CDS2 (Hepatitis C virus)" <220> <221> misc_feature <222> 76..86 <223> / note="Hybridization site for CDS2 (Hepatitis C virus)" <220> <221> misc_feature <222> 100..110 <223> / note="Hybridization site for CDS2 (Hepatitis C virus)" <220> <221> misc_feature <222> 124..134 <223> / note="Hybridization site for CDS2 (Hepatitis C virus)" <220> <221> misc_feature <222> 148..158 <223> / note="Hybridization site for CDS2 (Hepatitis C virus)" <220> <221> misc_feature <222> 172..182 <223> / note="Hybridization site for CDS2 (Hepatitis C virus)" <220> <221> misc_feature <222> 196..206 <223> / note="Hybridization site for CDS2 (Hepatitis C virus)" <220> <221> misc_feature <222> 220..230 <223> / note="Hybridization site for CDS2 (Hepatitis C virus)" <220> <221> misc_feature <222> 244..254 <223> / note="Hybridization site for CDS2 (Hepatitis C virus)" <220> <221> misc_feature <222> 266..278 <223> / note="Hybridization site for CDS2 (Hepatitis C virus)" <400> 6 cccccugggg cucugaugag gaaccucucu ggggucccca cagcgagucu ccuuggggcc 60 ccuagaauga aguucuuugg gguuucauag cacgguucuc cugggguuuu cauacgauug 120 ucucuugggg ucuugcgugu uuacccuuuu gggguccugc uaagggggcu uucuggggcc 180 uuucgaauaa gucuuuuugg ggccucguuu uaucacucuu cugggguucc cagccuuucc 240 cuucuugggg cuccuccgac caugccccuu gggguucuac ccuguaug 288 Sequence ID 7 <211> 10722 <212> RNA <213> Dengue virus <220> <221> misc_feature <222> 108..140 <223> / note=「cHP target area」 <220> <221> misc_feature <222> 10615..10646 <223> / note=「3UTR target area」 <400> 7 aguuguuagu cuacguggac cgacaaagac agauucuuug agggagcuaa gcucaacgua 60 guucuaacag uuuuuuaauu agagagcaga ucucugauga auaaccaacg gaaaaaggcg 120 aaaaacacgc cuuucaauau gcugaaacgc gagagaaacc gcgugucgac ugugcaacag 180 cugacaaaga gauucucacu uggaaugcug cagggacgag gaccauuaaa acuguucaug 240 gcccuggugg cguuccuucg uuuccuaaca aucccaccaa cagcagggau auugaagaga 300 uggggaacaa uuaaaaaauc aaaagcuauu aauguuuuga gaggguucag gaaagagauu 360 ggaaggaugc ugaacaucuu gaauaggaga cgcagaucug caggcaugau cauuaugcug 420 auuccaacag ugauggcguu ccauuuaacc acacguaacg gagaaccaca caugaucguc 480 agcagacaag agaaagggaa aagucuucug uuuaaaacag aggauggcgu gaacaugugu 540 acccucaugg ccauggaccu uggugaauug ugugaagaca caaucacgua caaguguccc 600 cuucucaggc agaaugagcc agaagacaua gacuguuggu gcaacucuac guccacgugg 660 guaacuuaug ggacguguac caccauggga gaacauagaa gagaaaaaag aucaguggca 720 cucguuccac augugggaau gggacuggag acacgaacug aaacauggau gucaucagaa 780 ggggccugga aacaugucca gagaauugaa acuuggaucu ugagacaucc aggcuucacc 840 augauggcag caauccuggc auacaccaua ggaacgac auuuccaaag agcccugauu 900 uucaucuuac ugachagcugu uucaucuuca augacaugc guugcauagg auugucaau 960 agagacuuug uggaagggu oucaggagga agcugggug acauagucuu agaacaugga 1020 agcuguguga cgacgauggc aaaaaaaaaaaaaaaaaaaaaaaacu gauaaaaaca 1080 gagccaaac agccugccac chuaggaag uacuguauag aggghaagcu aaccaaca 1140 acaagaau cucgcugcccc aacacaaggg gaacccagcc uaaaugaga gcaggacaaaa 1200 rocking gcaaaccuc caugguagac agaggagggg agaaggaug aggacuauuu 1260 ggaaagggg gcauugugac cugugcuaug oaaagaaaaaa 1320 guugugcaac cagaaacuu gaauacacc auugugauaa cuckoo agggaagg 1380 caugcagucg gaaugacac aggaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaching 1440 uccaucacag aagcagaauu guggagguuau ggcacuguca cauggagug cucuccaag 1500 acggggccucg acucauga gauggugug cugcagaugg aaaaaaagc uggcuggug 1560 cacaggcaau gguuccuga ccugccgua ccaugguugc ccggagcgga cacacaaggg 1620 ucaauugga oacagaaga gawaugguc acuuuaaaa auccccaugc gagaaacag 1680 gauguuguug uuuuaggauc ccaagaaggg gccaugcaca 1740 gaauccaa ugucaucagg aaacuuacuc ucacaggac aucucaagug caggcugaga 1800 auggacaagc uacagcuca aggaauguca uacucuaugu gcacaggaaauaaagou 1860 gogagaaaaaaaaaaaaaaaaaaaaaagaaaaugaaaaugaaaaaugaagggg 1920 gacggcucuc caugcaagou cccuuuugag auaauggaou uggaaaaag acaugucua 1980 ggucgccuga uacagucaa cccauugug achaaaag auagcccagu storm 2040 2100 cucaacuggu uuaagaaagg aaguucuauc ggccaaaugu uugagacaac aaugaggggg 2160 gcgaagaa uggccauuuu aggugacaca gccugggauu uuggauccuu gggagagug 2220 uuuacaucua uaggaaaggc ucuccaccaa gucuuuggag caaucuaugg agcugccuuc 2280 aguggguuu cauggacuau gaaaauccuc auaggaguca uuaucacaug gauaggaaug 2340 aauucacgca gcaccucacu gucugugaca cuaguauugg ugggaauugu gacacuguau 2400 uugggaguca uggugcaggc cgauaguggu ugcguuguga cguggaaaaa caaagaacug 2460 aaauguggca gugggauuuu caucacagac aacgugcaca cauggacaga acaauacaag 2520 uuccaaccag aaucccccuuc aaaacuagcu ucagcuaucc agaaagccca ugaagaggcgc 2580 auuuguggaa uccgcucagu aacaagacug gagaaucuga ugggaaaca aauaacacca 2640 gaauugaauc acaucuauuc agaaaugag gugaaugauaa cuuuauugac aggacauc 2700 aaaggaauca ugcaggcagg aaaacgaucu cugcggccuc agcccacuga gcugaaguau 2760 ucauggaaaa cauggggcaa agcaaaaug cucucacag agucucauaa ccagaccuuu 2820 cucauugaug gccccgaaac agcagaaugc cccaacaa auagagcuug gaauucgaug 2880 gagoogaag acuauggcuu uggauauuc accaccaaua uauggcuaaa auugaagaa 2940 aaacaggaug uauucgcga cucaaacuc augucagcgg cchauaaaga cacagagcc 3000 guccaugccg auuggguua uuggauaga agugcacuca augacacaug gagauagag 3060 aaagccucuu ucauugaagu uaaaaacugc cucuggccaa aucacacac ccucuggagc 3120 aaoggagugc oagaaaguga gaugauaauu ccaagaauc ucgcuggacc agugucucaa 3180 cacaacaua gaccaggcua cacaacacaa cauaacaggac cauggcaucu agguaagcuu 3240 gagauggacu ougauuucug ogauggaac ogauggaao cugcggaau 3300 agaggacccu auggugcugc aaccacugcc accuggaaac ucauacaga auggugcugc 3360 cgaucuugca cauaccacc gcuaagauac agaggugagg augggugcug guacgggaug 3420 gaaaacagac cauugaagga gaagagagag auuugguca acucuuggu cacagcugga 3480 caugggcagg ucgacacuu ucacuagga gucuuggga uggcauug ccuggagga 3540 augcuuagga cccgaguagg acgaaacau gcauacuac uaguugcagu uucuuuugug 3600 acauugauca cagggaacau guccuuuaga gaccuggga gagugauggu uaugguaggc 3660 gccacuauga cggaugacau agguaugggc gugacuuauc uugcccuacu agcagccuuc 3720 aaagucagac cauuuugc agcugacua cucuugagaa agcugaccuc cajaoug 3780 augaugacua quauaggaau uguacuccuc ucccagagca cacauaccaga gaccauucuu 3840 gaguugacug augcguuagc cuuaggcaug augguccuca aaauggugag aaauauggaa 3900 aaguaucaau uggcagugac uaucauggcu aucuugugcg ucccaaacgc agugauauua 3960 caaaacgcau ggaaagugag uugcacaaua uuggcagugg uguccguuuc cccacugcuc 4020 uuaacauccu cacagcaaaa aacagauugg auaccauuag cauugacgau caaaggucuc 4080 aauccaacag cuauuuuucu aacaacccuc ucaagaacca gcaagaaaag gagcuggcca 4140 uuaaaugagg cuaucauggc agucgggaug gugagcauuu uagccaguuc ucuccuaaaa 4200 aaugauauuc ccaugacagg accauuagug gcuggagggc uccucacugu gugcuacgug 4260 cucacuggac gaucggccga uuuggaacug gagagagcag ccgaugucaa augggaagac 4320 caggcagaga uaucaggaag caguccaauc cugucaauaa caauaucaga agaugguagc 4380 augucgauaa aaaaugaaga ggaagaacaa acacugacca uacucauuag aacaggauug 4440 cuggugaucu caggacuuuu uccuguauca auaccaauca cggcagcagc augguaccug 4500 roar agaacaacg ggccggagua roar scream scream 4560 ggaaggcug ooggaaaaaaaaaaaaaaaaggc aaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaggau 4620 ucccagaucg gagccggagu uaacaagaa ggaacaucc auacaugug gcaugucaca 4680 cguggcgcug uucuaugca uaaaggaaag aggauugaac caucaugggc ggacgucaag 4740 aaagaccuaa uaucauaugg aggaggcugg aggauagaag gagaoggaa gaggagaa 4800 gaaguccagg uauuggcacu ggagccugga aaaaauccaa gagccgucca acgaaaccu 4860 ggucuuuuca aaaccaacggc cggaacaaua ggugcuguau cucuggacuu ucuccugga 4920 acgucaggau cuccaauuau cgacaaaaaa ggaaaguug ugggucuuua ugguaauggu 4980 guguuacaa ggaguggagc auauugugagu gcuauagccc agacugaaa aagcauugaa 5040 cauggaccag agaacgaga ugacauuuuc cgaagagagaa gacugaccau cauggaccuc 5100 cacccaggag cgggaagac gagagauac cuccggcca uagucagaga agcuauaaaa 5160 cgggguuga gaacauaau cuggcccccc acuagaguug uggcagcuga auggaggaa 5220 gcccuuagag gacuuccaau aagauaccag accccagcca ucagagcuga gcacaccggg 5280 cgggagauug uggaccuaau gugucaugcc acauuuacca ugaggcugcu aucaccaguu 5340 agagugccaa acuacaaccu gauaucaug gacgaagccc auuucacaga cccagcaagu 5400 auagcagcua gaggauacau cucacucga guggagaugg gugaggcagc ugggauuuu 5460 augacagcca cucccccgggg aagcagagac accauuccuc agagcaugc accaaucaua 5520 gaugagaaa gagaauccc ugaacguucg uggauccg gaugaauug ggucacggau 5580 uuaaaggga agagugoog guucguucca aguaaaaag caggaauga uauagcagcu 5640 ugccugagga aaaauggaaa gaaagugaua caacucagua ggaagaccuu ugauucugag 5700 uaugucaaga cuagaaccaa ugauugggac uucguggua caacugacau uucagaaaug 5760 ggugccaauu ucaaggcuga gaggguuaua gaccccagac cugcaugaa accagucaua 5820 cuaacagaug gugaagagcg ggugauucug caggaccua ugccagugac ccacucuagu 5880 cagcacaaa gaagagggag aauaggaaga aauccaaaaa augagaauga ccaguacaua 5940 uacaugggg aaccucugga aaaugaugaa gacugugcac acuggaaaga agcuaaaaug 6000 cuccuagaua acaucaacac gccagaagga aucauuccua cauguucga accagagcgu 6060 gaaaaggugg augccauuga uggcgaauac cgcuugagag gagaagcaag gaaaaccuuu 6120 guagacuuaa ugagaagagg agaccuacca gucugguugg ccuacagagu ggcagcugaa 6180 ggcaucaacu acgcagacag aagguggugu uuugauggag ucaagaacaa ccaaaauccua 6240 gaaaaacg uggaguuga aaucuggaca aaagaagggg aaaggaagaa auugaaaccc 6300 agaugguugg augcuaggau cuauucugac ccacuggcgc uaaaagaauu uaaggaauuu 6360 gcagccggaa gaagucucu gacccugaac cuaaucacag aaauggguag gcucccaacc 6420 uucaugacuc agaaggcaag agacgcacug gacaacuuag cagugcugca cacggcugag 6480 cgagguggaa gggcguacaa ccaugcucuc aguugaacugc cggagacccu ggagacauug 6540 cuuuuacuga cacuucuggc uacagucacg ggagggaucu uuuuauucuu gaugagcgga 6600 aggggcauag ggagaugac ccugggaaug ugcugcauaa ucacggcuag cauccuccua 6660 ugguacgcac aaauacagcc acacuggua gcagcuucaa uaauacugga guuuuuucuc 6720 uaguuuugcu uauuccagaa ccugaaaaac agagaacacc ccaagacaac caacugaccu 6780 aggucau agccauccuc aguggugg ccgcaaccau ggcaaacgag auggguuucc 6840 wagaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaogg gaaaaaougc aaccaaa cccgaaaaack 6900 acauccugga cauagaucua cguccugcau cagcauggac gcuguaugcc guggccacacaa 6960 cauuuguuac accaauguug agacauagca uugaaauuc cucagugaau gugucccuaa 7020 cagcuauagc caccaagcc acaguguua ugggucucgg gaaggaugg cauuguca 7080 agauggacau cggaguuccc cunucucgcca uuggaugcua cucacaaguc aacccauaa 7140 cucucacagc agcucuuuuc uuauugguag cacauauugc caucauaggg ccaggacucc 7200 aaagcaaagc aaccagagaa gcucagaaaa gagcagcggc gggcaucaug aaaaacccaa 7260 cugucgaugg auaacagug auugaccuag auccaauacc uauugaucca auagoogaaa 7320 agcaguuggg acaaguaug cuccuagucc ucugcgugac acaaguauug augaugagga 7380 cuacaugggc ucugugugag gcuuuaaccu uagcuaccgg gccaucucc acouuguggg 7440 aaaggaaucc agggagguu ugggaacacua ccauugcggu gucaauggcu aacauuuuua 7500 gaggggaguua cuuggccgga gcuggacuuc ucuuuucuau uaugagaac acaaccaaca 7560 caagaagggg aacuggcaac auaggagaga cgcuuggga gaauggaa agccgauuga 7620 acgcauuggg aaaaagugaa uuccagaucu acaagaaaag uggaauccag gaaguggaua 7680 gaaccuuagc aaaagaaggc auuaaaagag gagaaacgga ccaucacgcu gugucgcgag 7740 gcucagcaaa acugagaugg uucguugaga gaacauggu cacaccagaa gggaaaguag 7800 uggacucgg uuguggcaga ggaggcuggu cauacuauug uggaggacua aagaauguaa 7860 gagagucaa aggccuaaca aaaggaggac caggacacga agaacccauc cccaugucaa 7920 cauaugggug gaaucuagug cgucuucaaaaa guggugauga cguuuuucuuc aucccgccag 7980 aaaaguguga cacauuauug ugugacauag gggagucauc accaaaucc acagugggaag 8040 caggacgaac acucagaguc cuuaacuuag uagaaaauug guugaacaac aacacuaau 8100 8160 aaaggaaaua uggagagcc uuagugaga auccacucuc acgaaacucc acacaugaga 8220 ugaacugggu auccaaugcu uccgggaaca uagugucauc aguugaacaug auuucaaagga 8280 uguuguucaa cagauuuaca augagauaca agaaagccac uaacgagccg gauguugacc 8340 ucggaagcgg aacccguaac aucgggauug aaagugagau accaaaccua gauauaauug 8400 ggaaaagaau agaaaaaau aagcaagagc augaaacauc auggcacuau gaccaagacc 8460 acccauacaa aacgugggca uaccauggua gcuaugaaac aaaacagacu ggaucagcau 8520 cauccauggu caacggagug gucaggcugc ugacaaaacc uugggacguc guccccaugg 8580 ugacacagau ggcaaugaca gacacgacuc cauuuggaca acagcgcguu uuuaaagaga 8640 aaguggacac gagaacccaa gaacgaaag aaggcacgaa gaacuaaug aaaauaacag 8700 cagaguggcu uuggaaaagaa uaggggaaga aaaagacacc caggaugugc accagagaag 8760 aauucacaag aaaggugaga agcaaugcag ccuugggggc cauauucacu gaugagaaca 8820 aguggaaguc ggcacgugag ccuguugaag auaguagguu uugggagcug guugacaagg 8880 aaaggaaucu ccaucuugaa ggaaagugug aacauugugu guacaacaug augggaaaaa 8940 gagagagaa gcuaggggaa uucggcaagg caaaaggcag cagagccaua uggucaaugu 9000 ggcuuggagc acgcuucuua gaguuugaag cccuaggauu cuuaaaugaa gaucacuggu 9060 ucuccagaga gaacucccug aguggagugg aaggaagg gcugcaaag cuagguuaca 9120 uucuaagaga cgugagcaag aaagagggag gagcaaugua ugccgaugac accgcaggau 9180 gggauacaag aaucacacua gaaccuaa aaaaugaaga aaugguaaca aaccacaugg 9240 aaggaaca caagaacua gccgaggcca uuuucaaacu aacguaccaa aacaaggugg 9300 9360 aaagagguag uggacaaguu ggcaccuaug gacucaauac uuucaccaau auggaagccc 9420 aacuaaucag acagauggag ggagaaggag ucuuuaaaag cauucagcac cuaacaauca 9480 cagagaaau cgcugugcaa aacugguuag caagugggg gcgcgaaagg uuaucaagaa 9540 uggcaucag ugggagaugau uguguuguga aaccuuuaga ugacagguuc cgaagcgcuu 9600 uaacagcucu aaaugacaug ggaaagauua ggaaagacau acaacaaugg gaaccuucaa 9660 9720 ugaaagacgg ucgcguacuc guuguuccau guagaaacca agaugaacug auuggcagag 9780 cccgaaucuc ccaaggagca ggguggucuu ugcgggagac ggccuguuug gggaagucuu 9840 acggcccaau guggagcuug auguacuucc acagacgcga ccucaggcug gcggcaaug 9900 cuauuugcuc ggcaguacca ucauuggg ggcaaccag ggcaacc ggcaguacca 9960 shake shake shake shake shake 10020 oocaaaaaa cccauggaug gagaaaa cuccagugga aucaugggag gaaucccau 10080 acuugggaa aagagagac caauggugcg gcucauugau uggguuaaaca agcagggcca 10140 ccuggggcaaa gaacauccaa gcagcaauaa aucaaguuag aucccuuaua ggcaugaag 10200 aaaacacaga uaacaugcca uccaaaaa gaucagag agagaggaa gagcaggag 10260 ucuguggua gaagcaaa cuacaugaa aaggcuag aaagucagguc gauaagcc 10320 auaguacgga aaaacuauug cuaccuguga gccccgucca aggacguaa aagaagocag 10380 gccaucauaa augccauagc uugaguaaac uuugcagccu guagcuccac cugagaaggu 10440 guaaaaaauc cgggaggcca caaaccaugg aagcuguacg cauggcguag uggacuagcg 10500 guuagaggag accccucccu uacaaaucgc agcaacaaug ggggcccaag gcgagaugaa 10560 gcuguagucu cgcuggaagg acuagagguu agaggagacc cccccgaaac aaaaaacagc 10620 auauugacgc ugggaaagac cagagauccu gcugucuccu cagcaucauu ccaggcacag 10680 aacgccagaa aauggaaugg ugcugugaa ucaacagguu cu 10722 Sequence ID 8 <211> 293 <212> RNA <213> Artificial arrangement <220> <223> Circular artificial sequence for 3UTR (dengue virus) (circ_dv_3utr) <220> <221> misc_feature <222> 7..38 <223> / note="Hybridization site for 3UTR (dengue virus)" <220> <221> misc_feature <222> 48..79 <223> / note="Hybridization site for 3UTR (dengue virus)" <220> <221> misc_feature <222> 89..120 <223> / note="Hybridization site for 3UTR (dengue virus)" <220> <221> misc_feature <222> 130..161 <223> / note="Hybridization site for 3UTR (dengue virus)" <220> <221> misc_feature <222> 171..202 <223> / note="Hybridization site for 3UTR (dengue virus)" <220> <221> misc_feature <222> 212..243 <223> / note="Hybridization site for 3UTR (dengue virus)" <220> <221> misc_feature <222> 253..284 <223> / note="Hybridization site for 3UTR (dengue virus)" <400> 8 gcgccgucuu uggucuuuuu uggcgucagu auguuguuuu uuaaccaucu uugguuuuuc 60 cuggcgucag ugugcuguua uaguaugguc ucugguuuu uuuagcguua gugugcuguu 120 aaagcuuucu cucuggucuu uuccaguguc aauaugcugu uuagugugcu ucuuugguuu 180 uucuuggugu uggugugcug uuucagcaag uucucugguc uuuucuggcg uugauguguu 240 guucgcuggc gaucuuuggu cuuucucggu gucgauaugu uguuaguggc cca 293 Sequence ID 9 <211> 300 <212> RNA <213> Artificial arrangement <220> <223> Circular artificial sequence for ChP (dengue virus) (circ_dv_cHP_v1) <220> <221> misc_feature <222> 7..39 <223> / note="Hybridization site for ChP (dengue virus)" <220> <221> misc_feature <222> 49..81 <223> / note="Hybridization site for ChP (dengue virus)" <220> <221> misc_feature <222> 91..123 <223> / note="Hybridization site for ChP (dengue virus)" <220> <221> misc_feature <222> 133..165 <223> / note="Hybridization site for ChP (dengue virus)" <220> <221> misc_feature <222> 175..207 <223> / note="Hybridization site for ChP (dengue virus)" <220> <221> misc_feature <222> 217..249 <223> / note="Hybridization site for ChP (dengue virus)" <220> <221> misc_feature <222> 259..291 <223> / note="Hybridization site for ChP (dengue virus)" <400> 9 gcgccgaugu uggggggugu guuuuuuguc uuuuuucguu gggguguaau guugagaggc 60 guguuuuuug cuuuuuuuug ugcaccuuau auauugggag guguguuuuu cgcuuuuuuc 120 cgucuggcug gcauauugg gggcguguuu uucguuuuuu uucguuacaa gcuaauauug 180 aagggugugu uuuucgccuu uuuccguguu aaagauaugu ugaaaggcgu guuuuucguu 240 uuuuuccgua aguuuuaaau auuggagggc guguuuuucg cuuuuuucug uagaccggaa 300 Sequence ID 10 <211> 294 <212> RNA <213> Artificial arrangement <220> <223> Circular artificial sequence for ChP2 (dengue virus) (circ_dv_cHP_v2) <220> <221> misc_feature <222> 7..39 <223> / note="Hybridization site for ChP2 (dengue virus)" <220> <221> misc_feature <222> 55..87 <223> / note="Hybridization site for ChP2 (dengue virus)" <220> <221> misc_feature <222> 103..135 <223> / note="Hybridization site for ChP2 (dengue virus)" <220> <221> misc_feature <222> 151..183 <223> / note="Hybridization site for ChP2 (dengue virus)" <220> <221> misc_feature <222> 199..231 <223> / note="Hybridization site for ChP2 (dengue virus)" <220> <221> misc_feature <222> 247..279 <223> / note="Hybridization site for ChP2 (dengue virus)" <400> 10 gcgccgaugu ugagaggugu guuuuuugcu uuuuuucguu uauuacucca acagauauug 60 agaggcgugu uuuucgcuuu uuuucguaga agguaauaga aaauauuggg gggcguguuu 120 uucgucuuuu uucgugacuc uuacgaguuu auguugaaag guguguuuuu cgcuuuuuuu 180 uguagggaac agucgcaaau auuggggggu guguuuuucg ccuuuuuucg uggcaccgua 240 auccgcaugu ugaaaggugu guuuuucguu uuuuuccguc aacucguucu uaua 294 Sequence ID 11 <211> 11840 <212> RNA <213> Chikungunya virus <220> <221> misc_feature <222> 14..48 <223> / note=「5UTR target area」 <220> <221> misc_feature <222> 11367..11399 <223> / note=「RSE target region」 <400> 11 auggcugcgu gagacacacg uagccuacca guuucuuacu gcucuacucu gcaaagcaag 60 agauuaauaa cccaucaugg auccugugua cguggacaua gacgcugaca gcgccuuuu 120 gaaggcccug caacgugcgu accccauguu ugagguggaa ccaaggcagg ucacaccgaa 180 ugaccaugcu aaugcuagag cguucucgca ucuagcuaua aaacuaauag agcaggaaau 240 ugaccccgac ucaaccaucc uggauaucgg cagugcgcca gcaaggagga ugaugucgga 300 caggaaguac cacugcgucu gcccgaugcg cagugcggaa gauccggaga gacucgccaa 360 uuaugcgaga aagcuagcau cugccgcagg aaaguccug gacagaaaca ucucuggaa 420 gaucggggac uuacaagcag uauggccgu gccagacacg gagacgccaa cauucugcuu 480 540 uguacacgca cccacgucgc uauaccacca ggcgauuaaa gggguccgag uggcguacug 600 gguuggguuc gacacaaccc cguucaugua caaugccaug gcgggugccu acccccuacaua 660 720. cucgacaaac ugggcagaug agcagguacu gaaggcuaag aacauaggau uauguucaac agaccugacg gagguagac gaggcaagu gucuauuaug agagggaaaa agcuaaaacc 840. snowflake snowflake 840. snowflake snowflake uagagcugg caccugccau cgguguucca uuuaaagggc aaacucagcu ucacaugccg 960. cugugauaca gugguuucgu guggggcua cgucguuaag agauaacga ugagcccagg 1020. ccuuuaugga aaaaccacag gguaugcggu aacccaccac gcagacggau uccugaugug caagacuacc gacacgguug acggcgaaag arugucauuc ucggugugca cauacgugcc ggcgaccauu ugugaucaaa ugaccggcau ccuugcuaca gaagucacgc cggaggaugc acagaagcug uugguggggc ugaaccagag aauagugguu aacggcagaa cgcaacggaa uacgaacacc augaaaaauu aucugcuucc cguggucgcc caagccuuca guaagugggc aaaggagugc cggaagaca uggagauga aaaacuccug ggggucagag aagaacacu 1320 gaccugcugc ugucuauggg caucaagaa gcagaaaca cacacggucu acagaggcc 1380 ugauacccag ucauucaga agcuucaggc cgaguuugac agcuuugugg uaccgagucu 1440 guggucgucc ggguuca ucccuuugag gacuagauc aaauggu uaagcaaggu 1500 gccaaaaacc gaccugaucc cauacagcgg agacccccga gaagcccggg acgcagaaaa 1560 agaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaacuacuacucgc? aggshaggaa gauguucagg ucgaaucga cgugghagg gagghagg gaggcggggcgc 1680 aggaauaaua gagacuccga gaggagcuau caaaguacu gcccaccaa cagaccacga 1740 cgugggag uaccugguac ucuccccgca gaccguacua cguagccaga accuguac 1800 gaucacgcu uuggcggagc aagugagac gugcacgcac aacggacgag cagggaggua 1860 1920 agacuuccag agucuaagcg aaagcgcaac gaugguguau aacgaaagag aguucguaaa 1980 cagaaagcua caccauauug cgaugcacgg accacgcccug aacaccgacg aagagucgua 2040 2100 augcuguaag aaaggaagaag ccgcaggacu gguacuggug ggcgacuuga cuaauccgcc 2160 cuaccacgaa uucgcauaug aagggcuaaa aauccgcccu gccugcccau acaaaauugc 2220 agucauagga gucuucggag uaccgggauc uggcaaguca gcuauuauca agaaccuagu 2280 uaccaggcag gaccugguga cuagcggaaa gaagaaaac ugccaagaaa ucaccaccga 2340 2400 gggaugcaac agaccagucg agguugugua cguagacgag gcguuugcgu gccacucugg 2460 aacgcuacuu gcuuugaucg ccuuggugag accaaggcag aaaguuguac uuugugguga 2520 2580 2640 ugugucaucg uugcauuacg aaggcaaaau gcgcacuacg aaugaguaca aaagccgau 2700 ugaguggac acuacaggcu caacaaaacc ugacccugga gaccucgugu uaacgugcuu 2760 cagagggugg guuaaacaac ugcaaauuga cuaucgugga uacgagguca ugacagcagc 2820 cgcaucccaa ggguuaacca gaaaaggagu uaacgcaguu agacaaaaag uaaaugaaaa 2880 2940 acugguaugg aagacacuuu ccggcgaccc guggauaaag acgcugcaga acccaccgaa 3000 aggaacuuc aaagcaacua uuaaggagug ggagguggag caugcaucaa uaauggcggg 3060 caucugcagu caccaauga ccuucgauac auuccaaau aaagccaacg uuuguugggc 3120 uaagagcuug gucccuaucc ucgaacagc ggggauaaa cuaugaua ggcagugguc 3180 ucagauaauu caagccuuca agagacaa agcauacuca ccugaaguag cccugaauuga 3240 auauaguacg cgcauguaug ggguggaucu agacaggggg cuauuuucua aaccguuggu 3300 gucuguguau uacgcggaua accacuggga uaauaggccu ggagggaaa uguucggauu 3360 uaaccccgag gcagcaucca oucuagaag aaaguaucca oucacaaag ggaaguggaa 3420 caucacaag cagaucugcg ugacuaccag gaggauaga gacuuuaacc cuaccaccaa 3480 caucauaccg gccacagga gacuaccaca cucauuagug gccgaacacc gcccaguaaa 3540 agggaaag auggaauggc ugguuaacha gauaaacggc caccacgugc uccuggucag 3600 uggcuauaac cuugcacugc cucuaagag cuugcacuugg guagcgccgu uagguguccg 3660 3720 ccuagugguc auaacaucc acacaccuuu ucgcauacac cauuaccaac agugcgugga 3780 ccacgcaaug aaacugcaaa ugcucggggg ugacucauug agacugcuca aaccgggcgg 3840 3900 auugggacgc aaguuuagau cgucuagagc guugaaacca ccauguguca ccagcaacac 3960 ugagauguuu uuccuauuca caacuuuuga caauggcaga agaauuuca caacuaugu 4020 caugaacaau caacugaaug cagcccuucgu aggacagguc acccgagcag gaugugcacc 4080 gucguaccgg guaaaacgca uggacaucgc gaaacgau gaagugcg uagucaacgc 4140 cgcuaacccu cgcggguuac cgggugrcgg uuuugcaag gcaguauaca aaaaauggcc 4200 ggaguccuu aagaacagug caacaccagu gggaaccgca aaaacaguua ugugcgguac 4260 guauccagua auccacgcug uuggaccaaa cuucucuaau uauucggagu cugaagggga 4320 ccgggaauug gcagcugccu aucgagaagu cgcaaaggaa guaacuaggc ugggaguaaa 4380 uaguguagcu auaccucucc ucuccacagg uguauacuca ggagggaaag acaggcugac 4440 ccagucacug aaccaccucu uuacagccau ggacucgacg gaugcagacg uggucaucua 4500 cugccgcgac aaagaauggg agaagaaaau aucugaggcc auacagaugc ggacccaagu 4560 agagcugcug gaugagcaca ucuccauaga cugcgauauu guucgcgugc acccugacag 4620 cagcuuggca ggcagaaaag gauacagcac cacggaaggc gcacuguacu cauaucuaga 4680 agggacccgu uuucaucaga cggcugugga uauggcggag auacauacua uguggccaaa 4740 gcaaacagag gccaaugagc aagucugccu auaugcccug ggggaaagua uugaaucgau 4800 caggcagaaa ugcccggugg augaugcaga cgcaucaucu ccccccaaaa cugucccgug 4860 ccuuugccgu uacgcuauga cuccagaacg cgucacccgg cucgcauga accacgucac 4920 aagcauaauu guguguucuu cguuuccccu cccaaguac aaaauagaag gagugcaaaa 4980 agucaaugc ucuaaggua ugcuauuuga ccacaacgug ccaucgcgcg uaaguccaag 5040 ggaauauaka ucuucccagg agcugcaca ggaggcgagu acaucacgu cacugacgca 5100 uagucaauuc gaccuagcg uugauggcga gauacugccc guccgucag accuggaugc 5160 ugacgcccca gcccugaac cgacquacuag cgacggggcg acacacgc ugcccacc 5220 aaccggaaac cuugcggccg ugucugauug gguaugagc acguaccug ucgcgccgcc 5280 cagagaagg cgagggaa accugacugu cauagugac gagagagag ggaauauaac 5340 accauggcu agcguccgau ucuuuagggc agagcugugu ccggucguac aagaaacagc 5400 ggagacgcgu ggacagcaa ugucucuuca ggcaccaccg aguaccgcca cggaaccgaa 5460 ucauccgccg aucuccuucg gagcaucaag cgagacguuc cccauuacau uuggggacuu 5520 caacgaagga gaaaucgaaa gcuugucuuc ugagcuacua acuuucggag acuucuuacc 5580 aggagaagug gaugacuuga cagacagcga cugguccacg ugcucagaca cggacgacga 5640 guuaugacua gacagggcag guggguauau auucucgucg gacaccgguc caggucauuu 5700 acaacagaag ucaguacgcc agucagugcu gccggugaac acccuggagg aaguccacga 5760 ggagaagugu uacccaccua agcuggauga agcaaaggag caacuauuac uuaagaaacu 5820 ccaggagagu gcauccaugg ccaacagaag cagguaucag ucgcgcaaag uagaaaacau 5880 gaaagcagca aucauccaga gacuaaagag aggcuguaga cuauacuuaa ugucagagac 5940 cccaaaaguc ccuacuuacc ggacuacaua uccggcgccu gugaacucgc cuccgaucaa 6000 cguccgauug uccaaucccg aguccgcagu ggcagcaugc aaugaguucu uagcuagaaa 6060 Cuauccaacu Gucucaucau Accaauuac Cgacgaguau Gaugcauauc Uagacauggu 6120 ggacgggucg gagaguugcc uggaccgagc gawuucau ccguxaaac uhaggagcua 6180 cccgaaacag cacgcuuacc acgcgccuc caucagaagc gcuguaccgu cccaucca 6240 gaacacacua cagaaguac uggcagcagc cacgaaaga aacugcaacg ucacacagau 6300 gagggaauua cccacuuugg acucagcagu auucaacgug gagugouuca aaaaauucgc 6360 augcaaccaa gauaacgggg aagaauuugc ugccagccccu auuaggauaa cacugagaa 6420 uuuagcaacc uuuguuacua aacuaaaagg gccaaaagca gcagcgcuau ucgcaaaaac 6480 ccauaaucua cugccacuac aggaaguacc auggauagg ucacaguag auugaaaag 6540 ggacguaaag gugacuccug guacaagca uacagaggaa agaccuaagg ugcagguauau 6600 acaggcggcu gaacccuugg cgacagcaua ccuauguggg auucacagag agcugguuag 6660 gaggcugaac gccguccucc uacccaaugu acauacacua uuugacaugu cugccgagga 6720 uuucgaugcc aucauagccg cacacuuuaa gccaggagac acuguuuugg aaacggacau 6780 agccuccuuu gauaagagcc aagaugauuc acuugcgcuu acugcuuuga ugcuguuaga 6840 ggauuuaggg guggaucacu cccugcugga cuugauagag gcugcuuucg gagagauuuc 6900 cagcugucac cuaccgacag guacgcgcuu caaguucggc gccaugauga aaucagguau 6960 guuccuaacu cuguucguca acacauuguu aaacaucacc aucgccagcc gagugcugga 7020 agaucgucug acaaaauccg cgugcgcggc cuucaucggc gacgacaaca uaauacaugg 7080 agucgucucc gaugaauuga uggcagccag augugccacu uggaugaaca uggaagugaa 7140 gaucauagau gcaguuguau ccuugaaagc cccuuacuuu uguggagggu uuauacugca 7200 cgauacugug acaggaacag cuugcagagu ggcagacccg cuaaaaaggc uuuuuaaacu 7260 gggcaaccg cuagcggcag gugacgac agagagau agagacgag cgcuggcuga 7320 cgaagugauc agauggcaac gacaggggcu auugaugag cuggagaag cgguauacuc 7380 uagguacgaa gugcagggua uaucagugu gguaaugucc auggccaccu uugcaagcuc 7440 cagauccaac uucgagaagc ucagaggacc cgucauaacu uuguacggcg guccuaaaua 7500 gguacgcacu acagcuaccu auuuugcaga agccgacagc aaguaucuaa acacuaauca 7560 gcuacaaugg aguucauccc aacccaacu uuuuacaaua ggagguacca gccucgaccc 7620 uggacuccgc gcccuacuau ccaagucauc agggcccagac cgcgcccuca gaggcaagcu 7680 gggcaacuug cccagcugau cucagcaguu aauaaacuga caugcgcgc gguaccccaa 7740 cagaagccac gcaggaaucg gagaauaag aaagcaaagc aaaaaaaaca ggcgccacaa 7800 aaaacacaa aucaaagaa gcagccaccu aaaaagaaac cggcucaaaa gaaaagaag 7860 ccgggccgca gagagaggau gugcaugaaa aucgaaaaug auuguauuuu cgaagucaag 7920 cacgaaggua agguaacagg uacgcgugc cugguggggg acaaaguaau gaaaccagca 7980 cacguaaagg ggaccaucga uaacgcggac cuggccaaac uggccuuuaa gcggucaucu 8040 aaguaugacc uugaaugcgc gcagauaccc gugcacauga aguccgacgc uucgaaguuc 8100 acccaugaga aaccggaggg guacuacaac uggcaccacg gagcaguaca guacucagga 8160 ggccgguuca ccaucccuac aggugcuggc aaccagggg acagcggcag accgaucuuc 8220 gacaacaagg gacgcguggu ggccauaguc uaggaggag quaaugaagg agcccguaca 8280 gcccucucgg uggugaccug gaauaaagac auugucacua aaaucacccc cgagggggcc 8340 gaagagugga gucuugccau cccaguuaug ugccuguugg caaacaccac guuccccugc 8400 ucccagcccc cuugcacgcc cugcugcuac gaaaaggaac cggaggaaac ccuacgcaug 8460 cuugaggaca acgucaugag accuggguac uaucagcugc uacaagcauc cuuaacaugu 8520 ucuccccacc gccagcgacg cagcaccaag gacaacuuca augucuauaa agccacaaga 8580 ccauacuuag cucacugucc cgacugugga gaagggcacu cgugccauag ucccguagca 8640 cuagaacgca ucagaaauga agcgacagac gggacgcuga aaauccaggu cuccuugcaa 8700 aucggaauaa agacggauga cagccacgau uggaccaagc ugcguuauau ggacaaccac 8760 augccagcag acgcagagag ggcggggcua uuuguaagaa caucagcacc guguacgauu 8820 acuggaacaa ugggacacuu cauccuggcc cgauguccaa aaggggaaac ucugacggug 8880 ggauucacug acaguaggaa gauuagucac ucauguacgc acccauuuca ccacgacccu 8940 ccugugauag gucgggaaaa auuccauucc cgaccgcagc acgguaaaga gcuaccuugc 9000 agcacguacg ugcagagcac cgccgcaacu accgaggaga uagagguaca caugccccca 9060 gacaccccug aucgcacauu aaugucacaa caguccggca acguaaagau cacagucaau 9120 ggccagacgg ugcgguacaa guguaauugc gguggcucaa augaaggacu aacacuaca 9180 gacaaguga uuaauaacug caagguugau caaugucaug ccgcggucac caaucacaaa 9240 aaguggcagu auaacucccc ucuggucccg cguaauugcug aacuuggga ccgaaaaagga 9300 aaaauucaca ucccguuucc gcuggcaaau guaacaugca gggugccuaa agcaaggaac 9360 9420 9480 aagagagaag ucgugcuaac cgugccgacu gaagggcucg aggucacgug gggcaacaac 9540 gagccguaua aguauuggcc gcaguuaucu aaaacggua cagcccaugg ccacccgcau 9600 9660 gccacguuca uacuccuguc gauggugggu auggcagcgg ggaugugcau gugugcacga 9720 cgcagaugca ucacaccgua ugaacugaca ccaggagcua ccgucccuuu ccugcuuagc 9780 cuauauugcu gcaucagaac agcuaaagcg gccacauacc aagaggcugc gauauaccug 9840 uggaacgagc agcaaccuuu guuuuggcua caagcccuua uuccgcuggc agcccugauu 9900 9960 9960 guaaugagcg ucggugccca cacugugagc gcguaacgaac aguaacagu gaucccgaac 10020 acgguggag uaccguauaa gacuguaguc aauagaccug gcuacagccc caugguauug 10080 gagauggaac uacugucagu cacuuuggag ccaacacuau cgcuugauua caucacgugc 10140 gaguacaaaa ccgucauccc gucuccguac gugaagugcu gcgguacagc agagucaag 10200 gacaaaaacc uaccugacua cagcuguaag gucuucaccg gcgucuaccc auuuaugugg 10260 ggcggcgccu acugcuucug cgacgcugaa aacacgcagu ugagcgaagc acacguggag 10320 aaguccgaau caugcaaaac agaauuugca ucagcauaca gggcucauac cgcaucugca 10380 ucagcuaagc uccgcguccu uuaccaagga aauaacauca cuguaacugc cuaugcaaac 10440 ggcgaccaug ccgucacagu uaaggacgcc aaauucauug uggggccaau gucuucagcc 10500 uggacaccuu ucgacaacaa aauuguggug uacaaaggug acgucuauaa cauggacuac 10560 ccgcccuuug gcgcaggaag accaggacaa uuuggcgaua uccaaagucg cacaccugag 10620 aguaaagacg ucuaugcuaa uacacaacug guacugcaga gaccggcugu ggguacggua 10680 cacgugccau acucucaggc accaucuggc uuuaaguauu ggcuaaaaga acgcggggcg 10740 ucgcugcagc acacagcacc auuuggcugc caaauagcaa caaacccggu aagagcggug 10800 aacugcgccg uagggaacau gcccaucucc aucgacauac cggaagcggc cuucacuagg 10860 gucgucgacg cgcccucuuu acggacaug ucgugcgagg uaccagccug acccaucc 10920 ucagacuuug ggggcgucgc cauuauuaaa uauugcagcca gcaagaaagg caagugugcg 10980 gugcauucga ugacuaacgc cgucacuauu cgggaagcug agauagagu ugaagggaau 11040 ucucagcugc aaaucucuuu cucgacggcc uuagccagcg ccgaauuccg cguacaaguc 11100 uguucuacac aaguacacug ugcagccgag ugccaccccc cgaaggacca cauagucaac 11160 uacccggcgu cacauaccac ccucgggguc caggacaucu ccgcuacggc gaugucaugg 11220 guggagaga ucacgggagg ugugggacug guguugcug ugccgcacu gaucuauc 11280 guggugcuau gcgugucguu cagcaggcac uaacuugaca auuaaguaug agaguauaug 11340 uguccccuaa gagacacacu guacauagca auauaaucuau agaucaagg gcuacgcaac 11400 cccugaauag uaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaacaaaaa 11460 uagguauacg uguccccuaa gagacacauu guauguaggu gauaaguaua gaucaaaggg 11520 ccgaauaacc ccugaauagu aacaaaauau gaaaaucaau aaaaaucaua aaauagaaaa 11580 accauaaaca gaaguaguuc aaagggcuau aaaaccccug aauaguaaca aaacauaaaa 11640 uuaauaaaaa ucaaaugaau accauaauug gcaaacggaa gagauguagg uacuuaagcu 11700 uccuaaaagc agccgaacuc acuuugagaa guaggcauag cauaccgaac ucuuccacga 11760 uucuccgaac ccacagggac guaggagaug uuauuuuguu uuuaauauuu caaaaaaaaa 11820 aaaaaaaaaa aaaaaaaaaa 11840 Sequence ID 12 <211> 294 <212> RNA <213> Artificial arrangement <220> <223> Circular artificial sequence for 5UTR (Chikungunya virus) (chikv_5utr1) <220> <221> misc_feature <222> 7..41 <223> / note="Hybridization site for 5UTR (Chikungunya virus)" <220> <221> misc_feature <222> 55..89 <223> / note="Hybridization site for 5UTR (Chikungunya virus)" <220> <221> misc_feature <222> 103..137 <223> / note="Hybridization site for 5UTR (Chikungunya virus)" <220> <221> misc_feature <222> 151..185 <223> / note="Hybridization site for 5UTR (Chikungunya virus)" <220> <221> misc_feature <222> 199..233 <223> / note="Hybridization site for 5UTR (Chikungunya virus)" <220> <221> misc_feature <222> 247..281 <223> / note="Hybridization site for 5UTR (Chikungunya virus)" <400> 12 gcgccgagua gagcagugag gaacuggugg guugcgugu uuuugucacu acguaguaga 60 guggaggaa auuggugggc ugcgugugu gcuuccagug ccaguagagc guggagagau 120 ugguagguua uguguguguu cucgcccgaa aguagagugg ugagggguug guagguuacg 180 uguguucaaa uaugacggag uagaguagug gggggcuggu ggguugugug uguucggaga 240 ccagacagua gaguaguaag agguuggugg gcuacgugu ugccacacca cccg 294 Sequence ID 13 <211> 314 <212> RNA <213> Artificial arrangement <220> <223> Circular artificial sequence for 5UTR (Chikungunya virus) (chikv_5utr2) <220> <221> misc_feature <222> 7..41 <223> / note="Hybridization site for 5UTR (Chikungunya virus)" <220> <221> misc_feature <222> 51..85 <223> / note="Hybridization site for 5UTR (Chikungunya virus)" <220> <221> misc_feature <222> 95..129 <223> / note="Hybridization site for 5UTR (Chikungunya virus)" <220> <221> misc_feature <222> 139..173 <223> / note="Hybridization site for 5UTR (Chikungunya virus)" <220> <221> misc_feature <222> 183..217 <223> / note="Hybridization site for 5UTR (Chikungunya virus)" <220> <221> misc_feature <222> 227..261 <223> / note="Hybridization site for 5UTR (Chikungunya virus)" <220> <221> misc_feature <222> 271..305 <223> / note="Hybridization site for 5UTR (Chikungunya virus)" <400> 13 gcgccgagua gaguggugg ggguugguag guugcgugu ucuuagcgcc aguagagcag 60 uaagggacug guaggcugcg uguguguac uuacaguaga gcggugagag acuggugggc 120 uaugugugu ccauccuuag uagaguggua gggaguuggu gggcugugu uguggggccu 180 auaguagagu agugggagau ugguagguua cguguguagc ugcacgagua gagcaguggg 240 aaguuggugg guugugugu ucuccgacga aguagaguag uaagaaauug guggguuaug 300 uguguaccug aguc 314 Sequence ID 14 <211> 288 <212> RNA <213> Artificial arrangement <220> <223> Circular artificial sequences for RSE (Chikungunya virus) (chikv_RSE1) <220> <221> misc_feature <222> 7..39 <223> / note="Hybridization site for RSE (Chikungunya virus)" <220> <221> misc_feature <222> 54..86 <223> / note="Hybridization site for RSE (Chikungunya virus)" <220> <221> misc_feature <222> 101..133 <223> / note="Hybridization site for RSE (Chikungunya virus)" <220> <221> misc_feature <222> 148..180 <223> / note="Hybridization site for RSE (Chikungunya virus)" <220> <221> misc_feature <222> 195..227 <223> / note="Hybridization site for RSE (Chikungunya virus)" <220> <221> misc_feature <222> 242..274 <223> / note="Hybridization site for RSE (Chikungunya virus)" <400> 14 gcgccguugc guggcucuuu ggucuauaga uuauuuguuu gcggguugcc cucuugcgug 60 guucuuuggu cuguggauug uuugcuugcc cgcgagcagg uuguguaguc cuuugaucug 120 uagguuguuu guuguucgua acacuucuug cguagcucuu ugaucuauag guuauuugcu 180 cuagguucga accguugcgu ggucuuuuga ucuauggguu guuuguucau caaaaccuuu 240 288 Sequence ID 15 <211> 288 <212> RNA <213> Artificial arrangement <220> <223> Circular artificial sequence for RSE2 (Chikungunya virus) (chikvRSE) <220> <221> misc_feature <222> 7..39 <223> / note="Hybridization site for RSE2 (Chikungunya virus)" <220> <221> misc_feature <222> 54..86 <223> / note="Hybridization site for RSE2 (Chikungunya virus)" <220> <221> misc_feature <222> 101..133 <223> / note="Hybridization site for RSE2 (Chikungunya virus)" <220> <221> misc_feature <222> 148..180 <223> / note="Hybridization site for RSE2 (Chikungunya virus)" <220> <221> misc_feature <222> 195..227 <223> / note="Hybridization site for RSE2 (Chikungunya virus)" <220> <221> misc_feature <222> 242..274 <223> / note="Hybridization site for RSE2 (Chikungunya virus)" <400> 15 gcgccguugc guggcucuuu ggucuauggg uuguuugcuu gcggguugcc cucuugugua 60 gcuuuuugau cuguggguua uuugcuugcc cgcgagcagg uugcguaguu cuuugauuug 120 uagguuguuu gcuguucgua acacuucuug cguaguuuuu ugaucuaugg auuguuugcu 180 cuagguucga accguugcgu agcucuuugg uuuauagauu auuugcucau caaaaccuuu 240 cuugugugu ccuuugaucu auagguuauu ugcuuccaac gacguacu 288 Sequence ID 16 <211> 620 <212> RNA <213> Artificial arrangement <220> <223> Broad-spectrum circular RNA for hepatitis C virus and dengue virus (DENV1 cHP_ HCV CDS2_T) <220> <221> misc_feature <222> 16..48 <223> / note="Hybridization site for CHP (dengue virus)" <220> <221> misc_feature <222> 58..90 <223> / note="Hybridization site for CHP (dengue virus)" <220> <221> misc_feature <222> 100..132 <223> / note="Hybridization site for CHP (dengue virus)" <220> <221> misc_feature <222> 142..174 <223> / note="Hybridization site for CHP (dengue virus)" <220> <221> misc_feature <222> 184..216 <223> / note="Hybridization site for CHP (dengue virus)" <220> <221> misc_feature <222> 226..258 <223> / note="Hybridization site for CHP (dengue virus)" <220> <221> misc_feature <222> 268..300 <223> / note="Hybridization site for CHP (dengue virus)" <220> <221> misc_feature <222> 330..340 <223> / note="Hybridization site for CDS2 (Hepatitis C virus)" <220> <221> misc_feature <222> 354..364 <223> / note="Hybridization site for CDS2 (Hepatitis C virus)" <220> <221> misc_feature <222> 378..388 <223> / note="Hybridization site for CDS2 (Hepatitis C virus)" <220> <221> misc_feature <222> 402..412 <223> / note="Hybridization site for CDS2 (Hepatitis C virus)" <220> <221> misc_feature <222> 426..436 <223> / note="Hybridization site for CDS2 (Hepatitis C virus)" <220> <221> misc_feature <222> 450..460 <223> / note="Hybridization site for CDS2 (Hepatitis C virus)" <220> <221> misc_feature <222> 474..484 <223> / note="Hybridization site for CDS2 (Hepatitis C virus)" <220> <221> misc_feature <222> 498..508 <223> / note="Hybridization site for CDS2 (Hepatitis C virus)" <220> <221> misc_feature <222> 522..532 <223> / note="Hybridization site for CDS2 (Hepatitis C virus)" <220> <221> misc_feature <222> 546..556 <223> / note="Hybridization site for CDS2 (Hepatitis C virus)" <220> <221> misc_feature <222> 570..580 <223> / note="Hybridization site for CDS2 (Hepatitis C virus)" <220> <221> misc_feature <222> 594..604 <223> / note="Hybridization site for CDS2 (Hepatitis C virus)" <400> 16 ggauccgcgg cgccgauguu ggggggugu uuuuuugucu uuuuucguug ggguguaaug 60 uugagaggcg uguuuuuugc uuuuuuuugu gcaccuuaua uauugggagg uguguuuuuc 120 gcuuuuuucc gucuggcugg cauauugggg ggcguguuuu ucguuuuuuu ucguuacaag 180 cuaauauuga agguguguu uuucgccuuu uuccguguua aagauauguu gaaaggcgug 240 uuuuucguuu uuuuccguaa guuuuaaaua uuggagggcg uguuuuucgc uuuuuucugu 300 agaccggaac cgggaauuca cucgagcccc cuggggcucu gaugaggaac cucucugggg 360 uccccacagc gagucuccuu ggggccccua gaugaaguu cuuugggguu ucauagcacg 420 guucuccugg gguuuucaua cgauugucuc uuggggucuu gcguguuuac ccuuuugggg 480 uccugcuaag ggggcuuucu ggggccuuuc gaauaagucu uuuuggggcc ucguuuuauc 540 acucuucugg gguucccagc cuuucccuuc uuggggcucc uccgaccaug ccccuugggg 600 uucuacccug uaugggaucc 620 Sequence ID 17 <211> 298 <212> RNA <213> Artificial arrangement <220> <223> Broad-spectrum circular RNA for hepatitis C virus and dengue virus (DENV1 cHP_ HCV CDS2_1) <220> <221> misc_feature <222> 7..22 <223> / note="Hybridization site for CDS2(2) (hepatitis C virus)" <220> <221> misc_feature <222> 35..67 <223> / note="Hybridization site for CHP (dengue virus)" <220> <221> misc_feature <222> 80..95 <223> / note="Hybridization site for CDS2(2) (hepatitis C virus)" <220> <221> misc_feature <222> 108..140 <223> / note="Hybridization site for CHP (dengue virus)" <220> <221> misc_feature <222> 153..168 <223> / note="Hybridization site for CDS2(2) (hepatitis C virus)" <220> <221> misc_feature <222> 181..213 <223> / note="Hybridization site for CHP (dengue virus)" <220> <221> misc_feature <222> 226..241 <223> / note="Hybridization site for CDS2(2) (hepatitis C virus)" <220> <221> misc_feature <222> 254..286 <223> / note="Hybridization site for CHP (dengue virus)" <400> 17 gcgccgccug gcuugggguu uucccaggac ucuaauauug gaaggcgugu uuuuugucuu 60 uuuucguguc cugccuccuc uuagccuggg guuuuacgac cgcaccuaug uugagaggug 120 uguuuuuugc cuuuuuucgu uugauacccu cauuuaaucu ggggcucuag gugcagcaaa 180 auguuggagg gcguguuuuu cguuuuuuuc uguccugcac gaucucccga uuugggguuc 240 ucguugguga cguauauuga gggguguguu uuucgcuuuu uuucguauug gacgucgu 298 Sequence ID 18 <211> 41 <212> RNA <213> Artificial arrangement <220> <223> An artificial array to illustrate an example of an operating mode shown in Figure 4, where the array does not have a specific value. <400> 18 aacgccaugc aagcgcgcag aucgagcugu gcgcuuuuuu u 41 Sequence ID 19 <211> 299 <212> RNA <213> Artificial arrangement <220> <223> circ_wnv_sIII_2 <400> 19 ugcaguuaga uaaacuuucc gguuugauuu ucucuucaaa agacaguucu ucgaacuucc 60 cggcuugguu uucuuuucaa aaauggggcc ccucaacuuc cuggucuggu uuucuccuua 120 aaaggcuaua cgaucaacuu ucuggccuga cuuucuucuu aaaaagcuau caucgcaacu 180 ucucggcuug acuuucucuu uaaaacccuc gguaaccaac uuuucggccu gauuuucuuc 240 ucaaaaguuu uggcgacuaa cuucuugguu uggcuuucuc cucaaaaaau auaaacagc 299 Sequence ID 20 <211> 11029 <212> RNA <213> West Nile virus <220> <223> Genome of West Nile virus <400> 20 aguaguucgc cugugugagc ugacaaacuu aguaguguuu gugaggauua acaacaauua 60 acacagugcg agcuguuucu uagcacgaag aucucgaugu cuaagaaacc aggagggccc 120 ggcaagagcc gggcugucaa uaugcuaaaa cgcggaugc cccgcguguu guccuugauu 180 ggacugaaga gggcuauguu gagccugauc gacggcaagg ggccaauacg auuuguguug 240 gcucucuugg cguucuucag guucacagca auugcuccga cccgagcagu gcuggaucga 300 uggagaggug ugaacaaaca aacagcgaug aaacaccuuc ugaguuuuaa gaaggaacua 360 gggaccuuga ccagugcuau caucggcgg agcucaaaac aaagaaag aggaaag 420 accggaauug cagucaugau uggccugauu gccagcguag gagcaguuac cucuac 480 uuccaaggga aggugaugau gacgwaaau gcuacugacg ucacagaugu caucacgaou 540 ccaacaugcug neighborhood ccuaugcauu cugagagca uggauguggg auacaugugc 600 gaugauacua ucacuuauga augcccagug cugucggcug guaugaucc agaagacauc 660 gacuguuggu gcacaaguc agcagucuac gagaguaug gagaugcac gacacacgc 720 cacucagac gcagucggag cucucaca gugcagacac acggagaaag cacucuagcg 780 aaaaaagg gggcuuggau ggacagcacc aaggccacaa gguauuuggu aaaaaacagaa 840 ucauggaucu ugaggaaccc uggauugcc cugguggcag ccgucauugg uuggaugcuu 900 gggagcaaca ccaugcagag aguuguuu gucgugcuau ugcuuuuggu ggccccagcu 960 uacagcuuca acugccuugg auugagcaac agagacuucu uggaaggagu gucuggagca 1020 acauggggg auuuggucu cgaggcgac agcugcguga cuaucauguc oaaggacaag 1080 ccuaccaucg augugaau gaugaauug gaggcggcca accuggcaga gguccgcagu 1140 uauugcuauu uggcuaccgu slow slow slow slow slow slow slow 1200 gaagcucaca augahaacg ugcugaccca gcuuuuugug ghagaagg agugguggac 1260 aggggcugggg gcaacggcug cggacuauuu ggcaaggaa gcauugacac augcgccaaa 1320 uuugccugcu cuaccaggc auaggaga accucuuga agagaauau caaguacgaa 1380 guggccauuu uuguccaugg accacuacu guggagucgc acggaacua cuccacacag 1440 guuggagcca cucaggcagg gagauucagc aucacuccug cggcgccuuc auacacacua 1500 aagcuoggag auauoggaga ggugacagug gacugugac cacggucagg gauugacacc 1560 aaugcauacu acgugaugac uguugggaaca aagacguucu ugguccaucg ugaguguuuc 1620 1680 uuaauggagu uugaggaacc acacgccacg aagcagucug uguaagcauu gggcucacaa 1740 gagggagcuc ugcaucaagc uuuggcugga gccauuccug uggaauuuuc aagcaacacu 1800 gucaaguuga cgucggguca uuugaagugu agagugaa uggaaaaaauu cgaguugaag 1860 ggaacaaccu auggcgucug uucaaagcu uucaaguuuc uugggacucc cgcagacaca 1920 ggucacggca cugguguguu ggaauugcag uacacuggca cggauggacc uugcaaaguu 1980 ccuaucucgu caguggcuuc auugaacgac cuaacgccag ugggcagauu ggucacuguc 2040 aacccuuuug uuucaguggc cacggccaac gcuaaggucc ugauugaauu ggaaccaccc 2100 uuugggacu cauacauagu ggugggcaga gggaacaac agaucaauca ccauuggcac 2160 aagucuggaa gcagcauugg caaagccuuu acaaccaccc ucaaaggagc gcagagacua 2220 gccgcucuag gagacacagc uugggacuuu ggaucaguug gagggguguu caccucaguu 2280 gggaaggcug uccaucaagu guucggagga gcauuccgcu cacuguucgg aggcaugucc 2340 uggauaacgc aaggauugcu gggggcucuc cuguugugga ugggcaucaa ugcucgugau 2400 agguccauag cucucacguu ucucgcaguu ggaggaguuc ugcucuuccu cuccgugaac 2460 gugcacgcug acacugggug ugccauagac aucagccggc aagagcugag auguggaagu 2520 ggaguguuca uacacaauga uguggaggcu uggauggacc gguacaagua uuacccugaa 2580 acgccacaag gccuagccaa gaucauucag aaagcucaua aggaaggagu gugcggucua 2640 cgaucaguuu ccagacugga gcaucaaaug ugggaagcag ugaaggacga gcugaacacu 2700 cuuuugaagg agaauggugu ggaccuuagu gucgugguug agaaacagga gggaauguac 2760 aagucagcac cuaacgccu caccgcacc acggaaaau uggaaauugg cuggaaggcc 2820 uggaggaaaaaaaaaaaaaaaaaaaaaaaaaaaaaooooo 2880 ccggagacca aggaaugucc gacucagaau cgcgcuugga auagcuuaga aguggaggau 2940 uuuggauuug gucucaccag uooooooooooooooooooooooooooooooooooooo backder storm 3000 gaaugugacu cgaagaucau uggaacggcu gaugagaca acuuggcgau ccacagugac 3060 cuguccuauu gauacguaag caggcucaau gauacgugga agcuugaag ggcaguucug 3120 ggugaaguca aucauguac guggccugag acgcauaccu uguggggcga uggauccuu 3180 gagagugacu ugauaauacc agucacacug gcgggaccac gaagcauca caacggaga 3240 ccuggguaca agacacaaa ccaggggccca ugggacgaag gccggguaga gauugacuuc 3300 gauuacugcc caggaacuac ggucacccug agugagagcu gcggacaccg 3360 acucgcacca ccacagagag cggaaaguug auaacagauu ggugcugcag gagcugcacc 3420 uuaccaccac ugcgcuacca aacugacagc ggcuguuggu augguaugga gaucagacca 3480 3540 auugacccuu uucaguuggg ccuucugguc guguuugg ccacccagga gguccuucgc 3600 aagaggugga cagccaagau cagcaugcca gcuauacuga uugcucugcu aguccuggug 3660 uuugggggca uuacuuacac ugauguguua cgcuauguca ucuugguggg ggcagcuuuc 3720 gcagaaucua auucgggagg agacguggua cacuuggcgc ucauggcgac cuucaagaua 3780 caaccagugu uuaugguggc aucguuucuc aaagcgagau ggaccaacca ggagaacauu 3840 uuguuguugu uggcggugu uuucuuucaa auggcuuauc acgaucccg ccaaauucug 3900 cucugggaga ucccugaugu guugaauuca cuggcgguag cuuggaugau acugagagcc 3960 auaacauuca caacgacauc aaacgugguu guuccgcugc uagcccugcu aacacccggg 4020 cugagacgcu ugaaucugga uguguacagg auacugcugu ugauggucgg aauaggcagc 4080 uugaucaggg agaagaggag ugcagcugca aaaaagaaag gagcaagucu gcuaugcuug 4140 gcucuagccu caacaggacu uuucaacccc augauccuug cugcuggacu gauugcaugu 4200 gaucccaacc guaaacgcgg auggcccgca acugaaguga ugacagcugu cggccuaaug 4260 uuugccaucg ucggagggcu ggcagagcuu gacauugacu ccauggccau uccaaugacu 4320 aucgcggggc ucauguuugc ugcuuuccgug auuucuggga aaucaacaga uauugggauu 4380 gagagaacgg cggacauuuc cugggaaagu gaugcagaaa uuacaggcuc gagcgaaaga 4440 guugaugugc ggcuugauga ugauggaaac uuccagcuca ugaaugaucc aggagcaccu 4500 uggagaauau ggaugcucag aauggucugu cucgcgauua gugcguacac ccccugggca 4560 aucuugcccu caguagougg auuuuggaua acucuccaau acacaaagag aggaggcgug 4620 uugugggaca cucccucacc aaaaaggggg acacgaccac cggcgucuac 4680 aggaucauga cucguggggcu gcucggcagu auucaagcag gagcggggcu gaugguugaa 4740 gguguuuucc acacccuuug gcauacaaaaggaggccg cubiugag cggagaggggc 4800 cgccuggacc cauacugggg cagugucaag gaggacgac uuuguuacgg aggacccugg 4860 aaauugcagc acaaguggaa cggggcaggau gaggggcaga ugauuguggu ggaaccuggc 4920 aaacaccuga aggaaauc 4980 ggggccguga cuuggacuu ccccacugga acaucaggcu caccaauagu ggaaaaac 5040 ggugaaguga uugggcuuua uggcaaugga gracouaugc ccaacggcuc auacauaagc 5100 gcgauagugc agggugaag gauggaag ccaaucccag ccggauucga accugagaug 5160 cugagaaaa aacagaucac ugaacuggau cuccaucccg gcgccgguaa aacaaggagg 5220 auucugccac agaucaucaa agaggccaua aacagaagac ugagaacagc cgugcuagca 5280 ccaaccaggg uuguggcugc ugagauggcu gaagcacuga gaggacugcc cauccgguac 5340 cagacauccg cagugcccag agaacauaau ggaaugaga uuguuguugu caugugucau 5400 5460 auggaugagg cucauuucac cgacccagcu agcauugcag caagagguua cauuuccaca 5520 aaggucgagc uaggggaggc ggcggcaaua uucaugacag ccacccccacc aggcacuuca 5580 5640 cguuggaacu cuggauacga auggaucaca gaauacaccg ggagaacggu uugguuugug 5700 ccuaguguca agauggggaa ugagauugcc cuuugccuac aacgugcugg aaagaaagua 5760 guccaauuga acagaaaguc guacgagacg gaguacccaa aauguaagaa cgaugauugg 5820 gacuuuguua ucacaacaga cauaucugaa auggggggcua acuucaaggc gagcagggug 5880 auugacagcc ggaaagugu gaaccaacc aucauaacag aaggaagagg gagagugauc 5940 cugggagaac caucugcagu gacagcagcu agugccgccc agagacgugg acguaucggu 6000 agaaauccgu cgcaaguugg ugaugaguac uguuauggg ggcacacgaa ugaagacgac 6060 ucgaacuucg cccauuggac ugaggcacga aucaugcugg acaacaucaa caugccaaac 6120 ggacugaucg cucaauucua ccaaccagag cgugagaagg uauauaccau ggaugggaa 6180 uaccggcuca gaggagaaga gagaaaaaac uuucuggaac uguugaggac ugcagauucug 6240 ccaguuuggc uggcuuaacaa gguugcagcg cugggagugu cauaccacga ccggaggugg 6300 ugcuuugaug gucuaaggac aaacacaauu uaagagaca acaacgaagu ggaagucauc 6360 acgaagcuug gugaaaggaa gaucugagg ccgcgcugga ougacgccag gguguacucg 6420 gaucaccagg cacuaaaggc guucaggac uucgccucgg gaaaacguuc ucagauaggg 6480 cucauugagg uucugggaa gaugccugag cacuucaugg ggagacaug gggagcacuu 6540 gaccaccagu accuugggc cugghaggag aaaggaa gaggcucacag aauggcccug 6600 gaggaacugc cagaugcucu ucagacaauu gccuugauug ccuuauugag ugugaugacc 6660 augggaguau ucuccuccu caugcagcgg aagggcauug gaagauagg uuugggaggc 6720 gcugucuugg gagucgcgac cuuuucugu uggaggcug aauguccagg acgaagauc 6780 gccggaaugu ugcugcucuc cucucuug augauugugc uaauuccuga gccagagaag 6840 caacguucgc agachagachaa caaccuagcc guguuccug uuuuguccug uuuuguucug 6900 agcgcagugg cagccaacga gauggguugg caguaaga ccaagaguga cauagcagu 6960 uuuuugggc aaagauuga ggucaggag aauuucagca ugggagu ccucuggac 7020 uugaggccgg cacagccug cucuguac gcugugacaa cucucug cucuccacug 7080 cuaaagcauu ugaucacguc agauacauc aacaccucau ugaccucau aaacguucag 7140 gcaagugcac uauucacacu cgcgcgaggc uucccuucg ucgauguugg agugucggcu 7200 cuccugcuag cagccggaug neighborhood cuccugcuca ccguuacggu aacagcggca 7260 accuccuuu uuugccacua ugccuacaug guucccgguu ggcaagcuga ggcaugcgc 7320 ucagcccagc ggcggacagc ggccggaauc augagaacg cuguagugga uggcaucgug 7380 gccacggacg ucccagaauu agagcgcacc acacccauca ogcagagaa agouggacag 7440 aucaugcuga aucaugcuguc guaguaguga accgucugu gagacagua 7500 cgagaagccg gaauuuugau cacggccgca gcggugacgc uuugggagaa uggagcaagc 7560 ucuguuugga acgcaacac ugccaucgga cucugccaca ucaugcgugg ggguuggug 7620 ucaugucuau ccauaacaug garicucaua aagaaaaaaaga aaaaaccagg acuaaaaaga 7680 ggggggcaaaggacgaaaaaaaaaaaggcaaaaaaaaaaggcaaca 7740 aaagagagu ucacuaggua ccgcaagag gccaucaucg aagucgaucg cucagcggca 7800 aaacacgcca ggaaagaagg caugucacu ggggcauc cagucucuag gggcacagca 7860 aaacugagau ggcuggucga acggagguuu cucgaccgg ucggaaagu gauugaccuu 7920 ggagooggaa gaggcgguug guguacuau auggcaaccc aaaaagagu ccaagaaguc 7980 agaggguaca aaagggcgg ucccggacau gagagcccc aacuagugca aaguauogga 8040 shakeshake shake shake shake shake shake shake shake 8100 gacccucc uuugugacau cggagagucc ucgucaagug cugagguug agagcauagg 8160 acgauucggg uccuugaaau gguugaggac uggcugcacc gagggccaag ggaauuuugc 8220 gugaaggugc ucugccccua caugccgaaa gucauagaga agauggagcu gcuccaacgc 8280 cgguaugggg ggggacuggu cagaaaccca cucucacgga auuccacgca cgagauguau 8340 ugggugaguc gagcuucagg caauguggua cauucaguga auugaccag ccaggugcuc 8400 cuaggaagaa uggaaaaaag gaccuggaag ggaccccaau acgaggaaga uguaaacuug 8460 ggaaguggaa ccagggcggu gggaaaaccc cugcucaacu cagacaccag uaaaaucaag 8520 aacaggauug aacgacucag gcgugaguac aguucgacgu ggcaccacga ugagaaccac 8580 ccauauagaa ccuggaacua ucacggcagu uaugauguga agcccacagg cuccgccagu 8640 ucgcugguca auggaguggu caggcuccuc ucaaaaccau gggacaccau cacgaauguu 8700 accaccaugg ccaugacuga cacuacuccc uucgggcagc agcgaguguu caaagagaag 8760 guggacacga acccuccuga accgccagaa ggagugagu accucuca cgagaccacc 8820 aacuggugu gggcguuuuuu ggccagagaa aaacguccca gaougugcuc ucgagaggaa 8880 uucauaagaa aggucaacag caugchaaaaugcca uguagaa uguuagaga gcagaauca 8940 uggaggaggcg ccagagaagc aguagagou shhaaaouuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuulyly’’  ggaggaggagggg 9000 cgcgaggcac aucugcgggg ggaagucac acuugcauuu acaucau gggaaagaga 9060 gagaaaaaac ccggagou cggaaggcc aagggaagca gaggauug guucaugugg 9120 cucggagcuc gcuuucugga guucgaggcu cuggguuuuc ucaugaga ccacuggcuu 9180 ggaagaaga acucaggagg aggugucgag ggcuugggcc uccaaaacu ggguuacauc 9240 cugcgugaag uggcaccccg gccuggggggc aagaucuaug cugaugacac agcuggcugg 9300 gandacccgca ucacgagagc ugacuuggaa aaugaagcua aggugcuuga gcugcuugau 9360 ggggacauc ggcgucuugc cagggccauc auugagcuca ccuaucguca caaaguugug aaagugaugc gcccggcugc aaagugaga accgucaugg auguuaucuc cagagaagau cagaggggga guggacaagu ugucaccuac gcccuaaaca cuuucacca ccuggccguc cagcugguga ggaugaugga aggggagga gugauuggcc cagaugaugu ggagaaacuc acaaaaggga aaggaccca agucaggacc uggcuguuug agaauggg agaaagacuc agccgcaugg cugucagugg agaugacugu gugguaaagc cccuggacga ucgcuuugcc accucgcucc acuuccuca ugcuauguca aagguucgca aagacaucca agaguggaa ccgucaacug gaugguauga uuggcagcag ccgucauuuu gcucaaacca uuucacuga uugaucauga aagauggaag aacacuggug guuccaugcc gaggacagga ugaauuggua ggcagagcuc gcauaucucc aggggccgga uggaacgucc gcgacacugc uugucuggcu aagucuuaug cccagaugug gcugcuucug uacuccaca gagagaccu gcggcucaug 10020 gccaacgcca uuugcuccgc ugucccugug auugggucc cuaccggaag aaccacgugg 10080 squeak shake shake shake shake shake shake 10140 goooooooooooooooooooooooooooooooooooo can to to to to this to touch here's how to goooooooooooooooooooooooooooooooooo gooooooooooooooooooooooooogperer 10200 guccauauu caggaaaacg agaggacauc ugguguggca gccugauugg cacaagagcc 10260 cgagccacgu gggcagaaaa cauccaggug gcuaucaacc aagucagagc auucaucgga 10320 gaugagago auguggaua caugagouca quaaagagau augagacac aacuuaggou 10380 gaggacacag uaugcauaaa auuuaauca uuuuaauaag acauauaag uauugcauaaaa 10440 Aoooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooggoogg 10500 agagaagucag gccgggaagu ucccgccacc ggaaguug oagcggugc ugccugcgac 10560 ucaaccccag gaggacuggg ugaacaaagc cgcgaaguga uccauguaag cccucagaac 10620 cguuucggaa ggaggacccc acauguugua acuucaaagc ccaaugucag accacgcuac 10680 ggcgugcuac ucugcggaga gugcagucug cgauagugcc ccaggaggac uggguuaaca 10740 aaggcaaacc aacgccccac gcggcccuag ccccgguaau gguguuaacc agggcgaaag 10800 gacuagaggu uagaggagac cccgcgguuu aaagugcacg gcccagccug gcugaagcug 10860 uaggucaggg gaaggacuag agguuagugg agaccccgug ccacaaaaca ccacaacaaa 10920 acagcauauu gacaccuggg auagacuagg agaucuucug cucugcacaa ccagccacac 10980 ggcacagugc gccgacaaug guggcuggug gugcgagaac acaggaucu 11029 Sequence No. 21 <211> 270 <212> RNA <213> Artificial sequence <220> <223> dchp_wsII_A <400> 21 ugcaguuaga uaauauuggg agguguguuu uuugccuuuu uccguagauc cucgcgaaac 60 uucuuggccu gguuuucuuu ucaaaagagu ccuuacguau auuggaggu guguuuuucg 120 cuuuuuuccg ucucggaaac gauaacuucc uggcuugauu uuuucuucaa aagcugcguu 180 aucuaauug agaggcgugu uuuucgucuu uuuccguucc aaccuggaga acuuuuuuggc 240 cugauuuuucu cuucaaaacc aggccguacc 270 sequence no. 22 <211> 270 <212> RNA <213> artificial arrangement <220> <223> dchp_wsII_B <400> 22 ugcaguuaga uaauauugga aggcguguuu uuugcuuuuu uccguagauc cuaacggaac 60 uuccuggccu gauuuuucuuc uuaaaagagu ccuuacguau auugaaaggu guguuuucg 120 ccuuuuuccg ucucggaaac gauaacuuuc cggccugguu uuuuccuuaa aagcugcguu 180 aucuaauug aggggugugu uuuuugucuu uuuccguucc aaccuggaga acuuccuggu 240 uuggcuuuuu ucuugaaacc aggccguacc 270 sequence number 23 <211> 270 <212> RNA <213> artificial arrangement <220> <223> dchp_wsII_C <400> 23 ugcaguuaga uaauauuggg agguguguuu uuugccuuuu uccguagauc cucgcgaaac 60 uucuuggccu gguuuucuuu ucaaaagagu ccuuacguau auuggaggu guguuuuucg 120 cuuuuuuccg ucucggaaac gauaacuucc uggcuugauu uuuucuucaa aagcugcguu 180 aucuaauug agaggcgugu uuuucgucuu uuuccguucc aaccuggaga acuucccggc 240 cugacuuuuu ccuuaaaacc aggccguacc 270 sequence number 24 <211> 299 <212> RNA <213> artificial arrangement <220> <223> circ_wnv_sIII_1 <400> 24 ugcaguuaga uaaacuuucu gguuugguuu ucuccucaaa agacaguucu ucgaacuucc 60 cgguuugauu uucucuuaa aaauggggcc ccucaacuuc uuggucugac uuucucuuca 120 aaaggcuaua cgaucaacuu cucggccuga uuuucuuuuc aaaaagcuau caucgcaacu 180 uccuggucug guuuucuucu caaaacccuc gguaaccaac uuuccggcuu gacuuucuuc 240 uuaaaacguu uugcgacaaa cuuuucggcu uggcuuucuc cuuaaaauau guaaacagc 299 Sequence ID 25 <211> 33 <212> RNA <213> Artificial arrangement <220> <223> Target sequence IRES Circ HCV1 <400> twenty five cuccgccaug aaucacuccc cugugaggaa cua 33 Sequence ID 26 <211> twenty four <212> RNA <213> Artificial arrangement <220> <223> Target sequence IRES Circ HCV2 <400> 26 ucucguagac cgugcaccau gagc 24 Sequence ID 27 <211> 28 <212> RNA <213> Artificial arrangement <220> <223> Target sequence cHP(CDS1)circ_hcv_cds1 <400> 27 ccaaaagaaa caccaaccgu cgcccaga 28 Sequence ID 28 <211> 16 <212> RNA <213> Artificial arrangement <220> <223> Target sequence CDS2 circ_hcv_cds2 <400> 28 ggggccccag g 16 Sequence ID 29 <211> 32 <212> RNA <213> Artificial arrangement <220> <223> Target sequence sHP(3'UTR)circ_dv_3utr <400> 29 aacagcauau ugacgcuggg aaagaccaga ga 32 Sequence ID 30 <211> 33 <212> RNA <213> Artificial arrangement <220> <223> Target sequence cHP1 / 2 circ_dv_cHP_v1 / 2 <400> 30 acggaaaaag gcgaaaaaaca cgccuuucaa uau 33 Sequence ID 31 <211> 34 <212> RNA <213> Artificial arrangement <220> <223> Target sequence Chikv_re <400> 31 gcugcuguaa aacguuggcu uuuuuagccg uaau 34 Sequence ID 32 <211> 294 <212> RNA <213> Artificial arrangement <220> <223> Broad-spectrum cyclic artificial sequences for DENV cHP and HCV CDS (circ_dv_cHP_v1_circ_hcv_cds2_2) <400> 32 gcgccgaugu ugaaaggugu guuuuucguu uuuuucuguc aaguguagga ucuucugggg 60 ucucgggccc cacgaccaau guuggggggc guguuuuucg cuuuuuucug ucaaaauaau 120 guugaccugg gguucuuuac ugaacucauu auguugggag gcguguuuuu ugucuuuuuu 180 uguuuuaucu acacccgucu gggguuuugu cggcgccacg acauguuggg ggguguguuu 240 uuugccuuuu uccgucucag ugcggcgccc uugggguucc cuuucgcauc acgu 294 Sequence ID 33 <211> 35 <212> RNA <213> Artificial arrangement <220> <223> Target sequence 5 UTR chikv5utr <400> 33 acacacguag ccuaccaguu ucuuacugcu cuacu 35 Sequence number 34 <211> 29903 <212> RNA <213> Coronaviridae <220> <223> NC_045512.2 Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) isolate Wuhan-Hu-1, complete genome <400> 34 auuaaagguu uauaccuucc cagguaacaa accaaccaac uuucgaucuc uuguagaucu 60 guucucuaaa cgaacuuuaa aaucugugug gcugucacuc ggcugcaugc uuagugcacu 120 cacgcaguau aauuaauaac uaauuacugu cguugacagg acacgaguaa cucgucuauc 180 uucugcaggc ugcuuacggu uucguccgug uugcagccga ucaucagcac aucuagguuu 240 cguccgggug ugaccgaaag guaagaugga gagccuuguc ccugguuuca acgagaaaac 300 acacguccaa cucaguuugc cuguuuuaca gguucgcgac gugcucguac guggcuuugg 360 agacuccgug gaggaggucu uaucagaggc acgucaacau cuuaaagaug gcacuugugg 420 cuuaguagaa guugaaaaag gcguuuugcc ucaacuuga cagcccuaug uguucaucaa 480 acguucggau gcucgaacug caccucaugg ucauguuaug guugagcugg uagcagaacu 540 cgaaggcauu caguacgguc guagugguga gacacuuggu guccuugucc cucauguggg 600 cgaaauacca guggcuuacc cgaagguucu ucuucguaag aacgguaaua aaggagcugg 660 uggccauagu uacggcgccg aucuaaaguc auuugacuua ggcgacgagc uuggcacuga 720 uccuuaugaa gauuuucaag aaaacuggaa cacuaacau agcaguggug uaacccguga 780 840 cccugauggc uacccucuug agugcauuaa agaccuucua gcacgugcug guaaagcuuc 900 augcacuuug uccgaacaac uggacuuuau ugacacuaag agggguguau acugcugccg 960 ugaacaugag caugaaauug cuugguacac ggaacguucu gaaagagcu augaauugca 1020 gacaccuuuu gaaauuaaau uggcaaagaa auuugacacc uucaaugggg aauguccaaa 1080 uuuuguauuu cccuuaaaau ccauaaucaa gacuauucaa ccaaggguug aaaagaaaaa 1140 gcuugauggc uuuaugggua gaauucgauc ugucuaucca guugcgucac caaaugaaug 1200 caaccaaaug ugccuuucaa cucucaugaa gugugaucau uguggugaaa cuucauggca 1260 gacgggcgau uuuguuaaag ccacuugcga auuuguggc acugagaauu ugacuaaaga 1320 aggugccacu acuugugguu acuuacccca aaaugcuguu guuaaaauuu auuguccagc 1380 augucacaau ucagaaguag gaccugagca uagucuugcc gaauaccaua augaaucugg 1440 cuugaaaacc auucuucgua aggguggucg cacuauugcc uuuggaggcu guguguucuc 1500 uuauguuggu ugccauaaca aguguccua uuggguucca cgugcuagcg cuaacauagg 1560 uuguaaccau acagguguug uuggagaagg uuccgaaggu cuuaaugaca accuucuuga 1620 aauacuccaa aaagagaaag ucaacaucaa uauuuguuggu gacuuuuaaac uaaauagaa 1680 gaucgccauu auuuggcau cuuuuuucugc uuccacaagu gcuuuuggg aaacugugaa 1740 agguuuggau uaaaagcau ucaaacaaau uguugaaucc ugugguaauu uuaaaguuac 1800 aaaaggaaaa gcuaaaaag gugccuggaa uauuggugaa cagaaaucaa uacugagucc 1860 ucuuuaugca uuugcaucag aggcugcucg uguuguacga ucaauuuuucu cccgcacucu 1920 ugaaacugcu caaaauucug ugcguuuuu acagaaggcc gcuauacaa uacuagaugg 1980 aauuuucacag uauucacuga gacucauuga ugcuaugaug uucacuucug auuuggcuac 2040 uaacaaucua guuguaaugg ccuacauuac aggugguguu guucaguuga cuucgcagug 2100 gcuacuaac aucuuuggca cuguuuuauga aaaacucaaa cccguccuug auuggcuuga 2160 agagaaguuu agaaaggug uagaguuucu uagagacggu ugggaaauug uuaaauuuau 2220 cucaaccugu gcuugugaaa uugucggugg acaaauuguc accugugcaa aggaaauuaa 2280 ggagaguguu cagacauucu uuaagcuugu aaauaaauuu uuggcuuugu gugcugacuc 2340 uaucauuau gguggagcua aacuuaaagc cuugaauuuua ggugaaacau uugucacgca 2400 cucaaaggga uuguacagaa aguguguuaa auccagagaa gaaacuggcc uacucaugcc 2460 ucuaaaagcc ccaaaagaaa uuaucuucuu agagggagaa acacuuccca cagaaguguu 2520 aacagaggaa guugucuuga aaacugguga uuuacaacca uuagaacaac cuacuaguga 2580 agcuguugaa gcuccauugg uugguacacc aguuuguauu aacgggcuua uguugcucga 2640 aaucaaagac acagaaaagu acugugcccu 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caccuaaacu guuaucaga 27660 caagagaag uucaagaacu uuacucucca auuuucuua uuguugcggc aauaguguuu 27720 auaacacuuu gcuucacacu caaaagaaag acagaaugau ugaacuuuca uuaauugacu 27780 ucuauuugug cuuuuuagcc uuucugcuau uccuuguuuu aauuaugcuu auuauucuuuu 27840 gguucucacu ugaacugcaa gaucauaaug aaacuuguca cgccuaaacg aacaugaaau 27900 uucuuguuuuu cuuaggaauc aucacaacug uagcugcauu ucaccaagaa ugaguuuac 27960 acuauguac ucaaucaa ccaauaguag uugaugaccc guguccuauu cacuuuuu 28020 cuaauggua uauuagagua ggagcuagaa aaucagcacc uuuaauugaa uugugcgugg 28080 augaggcugg uucuaauca cccauucagu acaucgauau cgguaauuau acaguuuccu 28140 guuuaccuuu uacaauuaau ugccaggaac cuaaauuggg uagucuugua gugcguuguu 28200 28260 cgaacaaacu aaaaugucug auaauggacc ccaaaucag cgaaaugcac cccgcauuac 28320 guuuggugga cccucagauu caacuggcag uaaccagaau gggagaacgca guggggcgcg 28380 aucaaaacaa cgucggcccc aagguuuacc caauaauacu gcgucuuggu ucaccgcucu 28440 cacucaacau ggcaaggaag accuuaaauu cccucgagga caagcguuc caauuaacac 28500 caauagcagu ccagaugacc aaauuggcua cuaccgaaga gcuaccagac gaauucgugg 28560 uggugacggu aaaaugaaag aucucagucc aagauggguau uucuacuacc uaggaacugg 28620 gccagaagcu ggacuuccu auggugcuaa caaagacggc aucauauggg uugcaacuga 28680 gggagccuug aauacaccaa aagaucacau uggcacccgc aauccugcua acaaugcugc 28740 aaucgugcua caacuuccuc aaagcaaac auugccaaaa ggcuucuacg cagaagggag 28800 cagaggcgg aguucaagccu cuucucguuc cucaucacgu agucgcaaca guucaagaaa 28860 uucaacucca ggcagcagua ggggaacuuc uccugcuaga auggcuggca auggcgguga 28920 ugcugcucuu gcuuugcugc ugcuugacag auugaaccag cuugagagca aaaugucugg 28980 uaaaggccaa caacaacaag gccaaacugu cacuaagaaa ucugcugcugcug aggcuucuaa 29040 gaagccucgg caaaaacgua cugccacuaa agcauacaau guaacacaag cuuucggcag 29100 acguggucca gaacaaaccc aaggaauuu ugggaccag gaacuaauca gacaaggaac 29160 ugauuacaaa cauuggccgc aaauugcaca auuugccccc agcgcuucag cguucuucgg 29220 aaugucgcgc auuggcaugg aagucacacc uucgggaacg ugguugaccu acacaggugc 29280 caucaaauug gaugacaaag auccaaauuu caagaucaa guacuuuugc ugaauaagca 29340 uauugacgca uacaaaacau ucccaccaac agagccuaaa areacaaaa agaagaggc 29400 ugaugaaacu caagccuuac cgcagagaca gaaaacag caaacuguga cucuucuucc 29460 ugcugcagau uuggaugauu ucuccaaaca augcaacaa uccaugagca gugcugacuc 29520 aacucaggcc uaaacucaug cagaccacac aaggcagaug ggcuauauaa acguuuucgc 29580 uuuuccguuu acgauauaua gucuacucuu gugcagaaug aauucucgua acuacauagc 29640 acaaguagau guaguuaacu uuaaucucac auagcaaucu uuaaucagug uguaacauua 29700 gggaggacuu gaagagcca ccacauuuuc accgaggcca cgcggaguac gaucgagugu 29760 acagugaaca augcuaggga gagcugccua uauggaaag cccuaaugug uaaaauuaau 29820 uuuaguagug cuauccccau gugauuuuaa uagcuucuua ggagaaugac aaaaaaaaaa 29880 aaaaaaaaaa aaaaaaaaaa aaa 29903 Sequence ID 35 <211> 33 <212> RNA <213> Artificial arrangement <220> <223> Target sequence RSE Circ chikvRSE <400> 35 agcaaauaau cuauagauca aagggcuacg caa 33 Sequence ID 36 <211> 294 <212> RNA <213> Artificial arrangement <220> <223> Artificial cyclic ARN 1; Target destruction structure contained in the SARS-CoV-2 3'UTR replication site <400> 36 ugcaguuaga uacugcuugu guuauguggu ugugaggguu uauuuuga uccugcuggu 60 uacuugucu guguaguuau gagaguuugu uuuggauacc uuuagccugc uuguguugu 120 ugguugcgag gauuuauucu gcucggaaac gauuuguuug uguuauguag uuguggggau 180 ucauucuggc ugcuuccucu uuauuugugu uguguaguua cgggaguucg uuuuguccca 240 cauagaguug cuugugcuau gugguuacgg ggauuuauuu ugccagggcc aaac 294 Sequence ID 37 <211> 29 <212> RNA <213> Artificial arrangement <220> <223> Target sequence SL III 3' UTR circ_wnv_slII_1 / 2 <400> 37 uuuugaggag aaagucaggc cgggaaguu 29 Sequence ID 38 <211> 294 <212> RNA <213> Artificial arrangement <220> <223> Artificial ring ARN 2; Target destruction structure contained in the SARS-CoV-2 3'UTR replication site <400> 38 ugcaguuaga uacugcuugu guuauguggu ugugaggguu uauuuuga uccugcuggu 60 uacuugucu guguaguuau gagaguuugu uuuggauacc uuuagccugc uuguguugu 120 ugguugcgag gauuuauucu gcucggaaac gauuuguuug uguuauguag uuguggggau 180 ucauucuggc ugcuuccucu uuguuugugu uguguaguua cggggguucg uuuuguccca 240 cauagaguua cuugugcuau guaguuauga ggauucauuu ugccagggcc aaac 294 Sequence ID 39 <211> 294 <212> RNA <213> Artificial arrangement <220> <223> Circ Chikv_re <400> 39 gcgccgauua uggcuaaaag agucaacguu uuguaguagc ugcggguugc ccucauuacg 60 guuaagaggg uuaauguuuu auagcagcug cccgcgagca ggauuauggu uggaaagguu 120 aacguuuugc agcagcguuc guaacacuuc auuauggcua aggaagccaa cguuuugugg 180 uagccuaggu ucgaaccgau uauggcuaga ggggucagcg uuuuauggua gccaucaaaa 240 ccuuucauua cggcugagag agccaauguu uuacaguagc uccaacgacg uacu 294 Sequence ID 40 <211> 294 <212> RNA <213> Artificial arrangement <220> <223> Artificial circular RNA 3; Target disruption structure contained in the SARS-CoV-2 3'UTR replication site <400> 40 ugcaguuaga uacugcuugu guuauguggu ugugaggguu uauuuuga uccugcuggu 60 uguuugugcu auguaguuac gaggguucau uuuggauacc uuuagccugc uuguguugug 120 ugguugcgag gauuuauucu gcucggaaac gauuuguuug uguuauguag uuguggggau 180 ucauucuggc ugcuuccucu cugcuugugc uguguaguua ugagaguuug uuuuguccca 240 cauagaguua uuuguguugu guaguugugg gaguuuguuu ugccagggcc aaac 294 Sequence ID 41 <211> 294 <212> RNA <213> Artificial arrangement <220> <223> Artificial circular RNA 4; Target disruption structure contained in the SARS-CoV-2 3'UTR replication site <400> 41 ugcaguuaga uauuguuugu gcuauugugu uguggggauu cauucugaug ucguauuguu 60 ugcuuguguu gugugguuac gggaauuugu uuugaaccucu uaguuguugu uugugcugug 120 uaguuguggg gguuuguucu gauguucuga ucuuuguuug uguuauguag uuacgaggau 180 uuauucuggu gcaacaaguu cuauuugugu uaugugguug cggggguucg uuuugcccgg 240 ccuucgcuua cuuguguugu guaguuguga ggauucguuu uggaugcauc gaac 294 Sequence ID 42 <211> 294 <212> RNA <213> Artificial arrangement <220> <223> Artificial circular RNA 5; Target disruption structure contained in the SARS-CoV-2 3'UTR replication site <400> 42 ugcaguuaga uacuauuugu gcuauguagu ugcggggguu uguuuugugg cuuucuuacc 60 uguuuguguu augugguugu gaggauuugu uuuggccgac ucguuccugc uugugcugug 120 uaguuguggg aguucauuuu guucagaguu agccuacuug uguuauguag uuaugagagu 180 ucguuuugga gagacgcaca cuguuugugu ugugugguua uggggauucg uuuugugggu 240 ccggcuccug uuugugcugu gugguuauga gaauuuguuc ugaagauuua uucc 294 Sequence ID 43 <211> 292 <212> RNA <213> Artificial arrangement <220> <223> Artificial circular RNA 1; Target disruption structure contained in SARS-CoV-2 5'UTR(SlII) <400> 43 ugcaguuaga uaagaucugc aggggguuga ggguugguug auccaggcug cuagaucuau 60 aagggguugg gaguugguuc uuuagcuauc ccagauuugu gagggguugg ggguugguua 120 ggcuuacagu uaagaucuau gggagguugg agguugguua ccgccgaguu auagauuuau 180 aggggaucgg aaguugguua aagcuagggc gaagaucugc gggggguugg aaguugguua 240 uacaacgaau uaagauuuau ggggggucgg ggguugguuu aguaguugga ug 292 Sequence ID 44 <211> 292 <212> RNA <213> Artificial arrangement <220> <223> Artificial circular RNA 2; Target disruption structure contained in SARS-CoV-2 5'UTR(SlII) <400> 44 ugcaguuaga uaggaucuau aagggguugg aaguugguua uguggucgcg cgaggucugu 60 aagagauuga aaguugguug ucacugacgu aaagauuuac gagagaucga ggguugguug 120 aaugucgguc ugggguuugu gggggguugg agguugguua uugguauaac ggaggucuau 180 gagagguuga ggguugguuc gcgauagaca ccgggucucc aggaggucgg aaguugguua 240 guugaacaga cuggguuuau ggggggucga gaguugguua uuuguuccga gg 292 Sequence ID 45 <211> 292 <212> RNA <213> Artificial arrangement <220> <223> Artificial circular RNA 3; Target disruption structure contained in SARS-CoV-2 5'UTR(SlII) <400> 45 ugcaguuaga uaagguuugc gagggauugg aaguugguug aaauguggaa ggggauuugc 60 gagagguuga aaguugguu uuauuaaagg uaggaucuac aagggauuga agguugguua 120 cccaguagcu caggguuuac aggaggucgg agguugguuu uucuggauag aaaggucuau 180 240 aaaacaagga acggauuuac aagggguugg ggguugguua uacuuagcgg ug 292 Sequence ID 46 <211> 282 <212> RNA <213> Artificial arrangement <220> <223> Artificial circular RNA 1; Target hybridization region: SARS-CoV-2 target A <400> 46 ugcaguuaga uauggugcug ugucgucguc uauucuaagu uugggagauc cugcuggugg 60 uauuauguua cugucuguuc uaaauuugag gauaccuuua gcuggugucg uguuauuguc 120 uguuuuggac uuaggcucgg aaacgauugg uacuguguca cugucuguuu uaggcuugaa 180 gcugcuuccu cuugguguua ugucaucguc uauuuugaac uuggguccca cauagagugg 240 uaccaugucg uugucuguuc uggauuuaaa ccagggccaa ac 282 Sequence ID 47 <211> 282 <212> RNA <213> Artificial arrangement <220> <223> Artificial circular RNA 2; Target hybridization region: SARS-CoV-2 target A <400> 47 ugcaguuaga uaugguacug ugucacuguc uguucugaac uugagcguuc acgucucugg 60 ugcuauguug ucgucuauuc uaaacuuaag aacuagguau aguggugucg uguuaucguc 120 uauuuugggc uugggggcuc cccuuggugg uaccauguca ccgucuauuc ugaauuuaaa 180 ucucccauag ugugguaucg uguugccguc uauuuuagac uuggaccacc gucuguuugg 240 uacuauguug cugucuguuu ugaacuugag aucagauuaa gg 282 Sequence ID 48 <211> 282 <212> RNA <213> Artificial arrangement <220> <223> Artificial circular RNA 3; Target hybridization region: SARS-CoV-2 target A <400> 48 ugcaguuaga uaugguaccg ugucgucguc uauuuuggau uuaaaagauc cugcuggugg 60 uauuaugucg uuguuuguuc ugaguuuaaa gauaccuuua gcugguacca uguuaccguc 120 uguucuaagu uuaaacucgg aaacgauugg uauuguguug ucguuuguuc uagauuuaaa 180 gcugcuuccu cuugguaucg ugucgccguc uauucugagc uuaaauccca cauagagugg 240 uacuauguug uugucuauuu ugaguuuaaa ccagggccaa ac 282 Sequence ID 49 <211> 282 <212> RNA <213> Artificial arrangement <220> <223> Artificial circular RNA 4; Target hybridization region: SARS-CoV-2 target A <400> 49 ugcaguuaga uaugguaccg ugucgccguu uguucugagc uuaaaguucu ugcgcucugg 60 uaucauguug cuguuuauuc uaggcuuaaa ucggccgucu gugguauug ugucguuguc 120 uguucugagu uuaaaucucu uucccggugg uacuguguug ucgucuauuc uggguuuaaa 180 acucgacccc gugguacca ugucaccguc uguuuuagac uuaaaguuac aagucgcugg 240 uauuauguug cugucuauuc uagauuuaaa auucccccag cc 282 Sequence ID 50 <211> 299 <212> RNA <213> Artificial arrangement <220> <223> Artificial circular RNA 1; Target hybridization region: SARS-CoV-2 target C <400> 50 ugcaguuaga uauagaggcc uugguggcag guuucuuggu gaagugagua uaguagaggu 60 cuuaguagca gguuuuuuag ugauguaacc uauguagaag ccucagcagc agauuucuug 120 gugggggguc aaggguaggg gucuuggugg uagauuuuuu gguggcacuu gaggaguagg 180 aguuucggcg guggauuucu uagugggcua ggaaauuugg gagcuuuggu gguagguuuu 240 uuggugaggu gggaggguug ggggcuucgg uagcggauuu uuuaguggaa cugaaagcg 299 Sequence ID 51 <211> 292 <212> RNA <213> Artificial arrangement <220> <223> Artificial circular RNA 2; Target hybridization region: SARS-CoV-2 target C <400> 51 ugcaguuaga uauaggaguu ucggcggcgg guuuuuuggu gaaaguugaa uuuagaaguu 60 ucgguggcgg guuucuuagu gagaaauuca gauagaaguu uuggcagugg auuucuuggu 120 gaggaauggg uuuggaagcc uuggagugg auuuuuuagu ggccacccuu gguagaagcu 180 uugguggag auuuuuuggu ggguaacuua auugggaguu uugguggcag guuuuuuagu 240 gauaggagac cguagggguu ucagcaguag auuucuuagu gggagacaga cu 292 Sequence ID 52 <211> 292 <212> RNA <213> Artificial arrangement <220> <223> Artificial circular RNA 3; Target hybridization region: SARS-CoV-2 target C <400> 52 ugcaguuaga uauaggaguc ucggcagcgg auuuuuuagu ggacaugacu uuuagaggcc 60 uuggagcgg auuucuuagu ggucuacagc gaugggaguu uuggcggugg guuuuuuggu 120 gaccucuaau guuggaggcu ucagcagcag guuuuuuagu gauaagagua gguaggggcc 180 uuaguagcag auuucuuggu gggauucgaa uuuagagguu uuaguaguag guuucuuagu 240 gaagggcuuu auuggggguu uuagcggcag auuuuuuggu ggauucgauc ug 292 Sequence ID 53 <211> 292 <212> RNA <213> Artificial arrangement <220> <223> Artificial circular RNA 4; Target hybridization region: SARS-CoV-2 target C <400> 53 ugcaguuaga uauggaaguu uuggcagugg guuucuuagu ggugggacuu gauagaaguc 60 uuagugguag guuucuuggu ggggcggagu guuggaggcu uugguagcgg guuuuuuagu 120 ggggcuaucu cgugggaguc uugguaguag auuucuuggu gaaggaugcg gguggaaguc 180 uuagcggcgg auuuuuuggu ggccaguuag uauggggguu uugguagcag guuuuuuggu 240 gauaguagac ucuggaggcu ucgguagcag auuuuuuagu gaguuauaaa gg 292 Sequence ID 54 <211> 270 <212> RNA <213> Artificial arrangement <220> <223> Artificial circular RNA 1; Target hybridization region: SARS-CoV-2 target D <400> 54 ugcaguuaga uaaaaccgua agcagccugu agaagguaga cgaguccaga cguacaaacc 60 guaggcagcu ugcagaagau agacgaagaa cgaguacuaa accgugggca guuugcgggg 120 gauagacgaa gcuuggagac uaaaccguaa guagucugu ggggauggac gaauuucugg 180 caauaaaccg uagguaguuu gcagagggua gacgagcucg uagcuagaaa ccgugaguag 240 ccuguagagg auggacgacu gagaccucaa 270 Sequence ID 55 <211> 270 <212> RNA <213> Artificial arrangement <220> <223> Artificial circular RNA 2; Target hybridization region: SARS-CoV-2 target D <400> 55 ugcaguuaga uaaaaccgua ggcagccugu aggagguaga cgaguuuucu cacggaaacc 60 guggguagcu uguaggagau agacgaguug aggaccagaa accguaagug guuugcagga 120 gauggacgag uuugggcagu uaaaccguga gcagucugca ggggguagac gaauguucgg 180 agcaaaaccg ugaguaguuu guagaaggug gacgagagga gaguaggaaa ccguaagcag 240 ucuguagggg auggacgauu uauaaucagu 270 Sequence ID 56 <211> 270 <212> RNA <213> Artificial arrangement <220> <223> Artificial circular RNA 3; Target hybridization region: SARS-CoV-2 target D <400> 56 ugcaguuaga uaaagccgug agcggcuugu ggaagauaga ugaaaggauu ggaaaagacc 60 guaagcagcu uguagaagau gggugacaag uaauggaaaa auguaagug gucugcggaa 120 ggugggugga uaaggaacuu gagaucguag guaguuugua gaggguaggu gggacuaugg 180 ggaugaacug ugggcggucu gcaggaggug gacgagagag guacauaaga ccguaaguag 240 uuugggggg augggcgaug uauugcacac 270 Sequence ID 57 <211> 270 <212> RNA <213> Artificial arrangement <220> <223> Artificial circular RNA 4; Target hybridization region: SARS-CoV-2 target D <400> 57 ugcaguuaga uaaagccgug agcggcuugu ggaagauaga ugaaaggauu ggaaaagacc 60 guaagcagcu uguagaagau gggugacaag uaauggaaaa auguaagug gucugcggaa 120 ggugggugga uaaggaacuu gagaucguag guaguuugua gaggguaggu gggacuaugg 180 ggaugaacug ugggcggucu gcaggaggug gacgagagag guacauaaag ccguaaguag 240 cuuguagggg auagauggug uauugcacac 270 Sequence ID 58 <211> 27 <212> RNA <213> Artificial arrangement <220> <223> The SARS-CoV-2 target hybridization region -5'UTR SLII is included in the SLII. <400> 58 aaccaacuuu cgaucucuug uagaucu 27 Sequence ID 59 <211> 33 <212> RNA <213> Artificial arrangement <220> <223> SARS-CoV-2 target hybridization region - Target A <400> 59 uuuaaguuua gaauagacgg ugacauggua cca 33 Sequence ID 60 <211> 30 <212> RNA <213> Artificial arrangement <220> <223> SARS-CoV-2 target hybridization region - Target C <400> 60 ucacuaagaa aucugcugcu gaggcuucua 30 Sequence ID 61 <211> 31 <212> RNA <213> Artificial arrangement <220> <223> SARS-CoV-2 target hybridization region - Target D <400> 61 ucgucuaucu ucugcaggcu gcuuacgguu u 31 Sequence ID 62 <211> 35 <212> RNA <213> Artificial arrangement <220> <223> SARS-CoV-2 target hybridization region - comprised in 3UTR Replication Site <400> 62 cagaaugaau ucucguaacu acauagcaca aguag 35 Sequence ID 63 <211> 289 <212> RNA <213> Artificial arrangement <220> <223> Artificial circular RNA (circWD1-in vitro) for WNV and DENV <400> 63 ugcaguuaga uaugcaguua gauaauauug ggaggugugu uuuuugccuu uuuccguaga 60 uccucgcgaa acuucuuggc cugguuuucu uuucaaaaga guccuuacgu auauuggagg 120 guguguuuuu cgcuuuuuuc cgucucggaa acgauaacuu ccuggcuuga uuuuuucuuc 180 aaaagcugcg uuaucuauau ugagaggcgu guuuuucguc uuuuuccguu ccaaccugga 240 gaacuuuuug gccugauuuu cucuucaaaa ccaggccgua ccgacaguc 289 SEQ ID NO: 64 <211> 307 <212> RNA <213> Artificial sequence <220> <223> HCV artificial circular RNA (circHCV-in vitro) <400> 64 ugcaguuaga uacccccugg ggcucugaug aggaaccucu cugggguccc cacagcgagu 60 cuccuugggg ccccuagaau gaaguucuuu gggguuucau agcacgguuc uccugggguu 120 uucauacgau ugucucuugg ggucuugcgu guuuacccuu uugggguccu gcuaaggggg 180 cuuucugggg ccuuucgaau aagucuuuuu ggggccucgu uuuaucacuc uucugggguu 240 cccagccuu cccuucuugg ggcuccuccg accaugcccc uuggguucu acccuguaug 300 gacaguc 307 > அக்கை CCCGGTTCCGCACTACTTGTGCTGTGTAGTTACGAGGATTCGTTCTGACCGTGTCTTCGCTATTTGTGCTATGTAGTTAT GGGAGTTTATTCTGCTGCTCGTGGACCTACTTGTGTTATGTAGTTGTGGGAGTTCGTCTGGGGCAGTTCGCTCTATTGTGCTGTGTGGTTGCGGGGTTCATTTTGATCCCGATAAGCCTACTTGTGTTGTGTAGTTATGAGGGTTTGTTTTGACCATACGACAGCTACTTGTGCTATGTGGTTGTGAGGATTCATTCTGGACAGTC > அக்கை CCCGGTTCCGCAAGATTTGTAGGAGGTCGAAAGTTGGTTTACAACGCTACTGGGGTCTACAGGAGGTTGAAGGTTGGTTGTCATAATGCAGTGGATCATCAGGGGGTCGAGAGTTGGTTGCCGCCATTCACAGGTCTGCAGGGGTCGGAAGTTGGTTAGCAGATGTTGAAGGTCTACGAGAGATTGGGGGTTGGTTAGGCTCGGAAAGAAAGATTTATAGGGGGTTGGAGGTTGTTCCAGTCAGTTTACGGATCTACAAGGGGTTGGGAGTTGGTTGACAGTC > அக்கை CCCGGTTCCGCAAGATTTGTAGGAGGTCGAAAGTTGGTTTACAACGCTACTGGGGTCTACAGGAGGTTGAAGGTTGGTTGTCATAATGCAGTGGATCATCAGGGGGTCGAGAGTTGGTTGCCGCCATTCACAGGGTCTGCGAGAGGTCGGGAAGTTGGTTAGCAGATGTTGAAGGTCTACGAGAGATTGGGGGTTGGTTAGGCTCGGGAAGAAAGATTTATAGGGGGTTGGAGGTTGGTTCCAGTCAGTTTACGGATCTACAAGGGGTTGGGAGTTGGTTGACAGTC > அக்கை CCCGGTTCCGCATGGTATTGTGTTACCGTCTATTTTAAGCTTAAGGCCCCCAAACTAGTTGGTGCCGTGTTGCCGTCTGTTCTAAGCTTAAGCTCGATCCCGTGTTTGCTATGTCACCGTCTATTCTAGACTTAGGCCGTCACTATCATTTGGTGTTGTGTTATTGTCTATTTTGGACTTGAACATGATTACACAACTGGTATCGTGTTGTCTGTTTTTAGACTTAGGTCGCTTTTCTTTCCTGGTACCATGTCGCCGTCTTGTTTGGGCTTGAGGACAGTC >> CCCGGTTCCGCATGGTATTATGTTGCCGTCTATTCTGGACTTAAACATTCCGTCATAGCTGGTGTCGTGTTATCGTCTATTCTAAACTTGAGGTCACAGTCGAACCTGGTGCTATGTCGTTGTCTGTTCTGGGTTTAAGCTACTTTTTCATGATGGTACTGTGTTACTGTCTGTTCTAGACTTGGGCGAGCAGAGATCTTTGGTACTATGTCACCGTCTATTTAAGCTTGGGATTAAAGCACCTGGTGGTACCGTGTCGCTGTCTATTTTGGCTTAGAGACAGTC > அக்கை CCCGGTTCCGCATGGTATTATGTTATTGTTTGTTCTGGGCTTAAAGAAGTACCATTAGGTGGTATCGTGTTGTTGTTTATTCTAGATTTAAAGACCTGGGGGCATCTGGTACTGTGTTGTCGTCTTATTTAGACTTAAAGTGGGCGATCGAGTTGGTACCATGTCATCGTTTTATTTAGGCTTAAACACAGATTCGCCTCTGGTACTATGTCGCCGTTTATTTTGGTTAAATATGCGTGGAGCAATGGTATCATGTTACCGTCTATTTTGAACTTAAAGACAGTC > அக்கை CCCGGTTCCGCATAGGGGCCTTGGCAGTAGGTTTTTTAGTGGAGTAGCCATTTAGGGGTTTCAGTGGTAGATTTCTTAGTGGAACCATATTGTAGGAGTCTCGGGTAGTGATTTCTTGGTGAATCGCTTGGCTGGAAGCCTTAGTAGCAGGTTTTTTGGTGGCAGCTAAGCATAGAGGCTCCGGTAGTAGATTTTTTAGTGATCGTAGAGGATAGGGGCTTTGCAGCAGATTTTTTGGTGGGAGATTGCCTTAGGAGCCTCGCAGGTTTCTTGGTGAGACAGTC > அக்கை CCCGGTTCCGCATAGGAGCCTTAGCGGTAGGTTTCTTAGTGAACTATTATTTTAGAAGCTTTAGTAGCGGATTTTTTGGTGGTTTTTATAACTGGAGGTTTCAGTGGCAGGTTTTTTTAGTGGAGGAGTGAGCTGGAAGTCTCGGTAGCAGGTTTTTTGGTGATGGCATTGACTGGAAGTCTTGGCGGTAGATTTCTTGGTGAGTCCACCGATGGAAGTTTCAGCGGCGGTTTCTTGGTGGTGAATGAACTTAGGGGCTTCAGCGGCGATTTCTTAGTGGGACAGTC sequence no. 73 5′-FAM-ACCCCGCATTACGTTTGGTGGACC-BHQ1-3′ Sequence ID 74 5′-GACCCCAAAATCAGCGAAAT-3′ Sequence ID 75 5′-TCTGGTTACTGCCAGTTGAATCTG-3 Target 1: IRES1 (Domain II) Sequence ID 76, TIFF2026102653000011.tif10170 Target 2: IRES 2 (Domain IV) Sequence ID 77 TIFF2026102653000012.tif6170 target 3: CDS 1(cHP) Sequence ID 78; TIFF2026102653000013.tif5170 target 4: CDS2(SL427) Sequence ID 79; TIFF2026102653000014.tif11170TIFF2026102653000015.tif6170 target 1:5'UTR: Sequence ID 80, TIFF2026102653000016.tif10170 Target 2: Iterative Array Elements: Sequence ID 81, TIFF2026102653000017.tif6170 Target 3: Re-coding element: Sequence ID 82, TIFF2026102653000018.tif11170TIFF2026102653000019.tif5170 Target 1:SLII: Sequence ID 83, TIFF2026102653000020.tif5170TIFF2026102653000021.tif5170 Target 1: Completely conserved replication sites in pangolins and RatG13 Sequence ID 84, TIFF2026102653000022.tif4170 Target 3: Target A Sequence ID 85, TIFF2026102653000023.tif4170 Target 4: Target C Sequence ID 86 TIFF2026102653000024.tif5170 Target 5: Target D Sequence ID 87, TIFF2026102653000025.tif6170>Array 88 CCCGGTTCCGCACTACTTGTGTTATGTAGTTACGAGGATTCGTTTTGCTCTTTTCTCCCTTATTTGTGTTGTGTAGTTATGGGAGTTTGTTCTGGCGTGGTACGTCTTACTTGTGTTGTGTGGTTATGAGAGTTCATTCTGTAT CACCACTAGCTGCTTGTGCTGTGTAGTTATGAGGATTTATTCTGGAATACCGTTCCCTGTTTGTGCTGTGTGGTTATGGGAGTTCGTTCTGTGAGGTGTTCCACTGCTTGTGCTATGTGGTTACGAGAATTTGTTTTGGACAGTC >Sequence code 89 CCCGGTTCCGCACTACTTGTGCTGTGTAGTTACGAGGATTCGTTCTGACCGTGTCTTCGCTATTTGTGCTATGTAGTTATGGGAGTTTATTCTGCAACCAGTGGACCTACTTGTGTTATGTAGTTGTGGGAGTTCGTTCTGGGG CAGTTCGCTCTATTTGTGCTGTGTGGTTGCGGGGGTTCATTTTGATCCCGATAAGCCTACTTGTGTTGTGTAGTTATGAGGGTTTGTTTTGACCATACGACAGCTACTTGTGCTATGTGGTTGTGAGGATTCATTCTGGACAGTC [Examples]

[0249] Example 1: The inventors have identified four circRNAs (circ_hcv_ires1 (SEQ ID NO: 2), circ_hcv_cds1 (SEQ ID NO: 4), circ_hcv_cds2 (SEQ ID NO: 6), y Circ_hcv_combo1 (SEQ ID NO: 5) was designed and tested against two different regions of the HCV genome (see SEQ ID NO: 1), as previously disclosed in the section on circRNA generation. Circ_hcv_ires1 contains seven 33-nucleotide hybridization sites targeting sequences within IRES elements. Circ_hcv_cds1 contains eight 28-nucleotide hybridization sites targeting sequences within coding regions. Circ_hcv_cds2 contains twelve hybridization sites targeting sequences within coding regions. Circ_hcv_combo1 contains three hybridization sites per target region (IRES1 (SEQ ID NO: 25), IRES2 (SEQ ID NO: 26), and cHP (CDS1) (SEQ ID NO: 27)). Target regions in the HCV genome are shown in Figure 6 and SEQ ID NO: 1. Both IRES1 (domain IV) and IRES2 (domain V) target disruption structures were described in C Romero-Lopez et al. 2007 (Cell Mol Life). Sci.2007 Nov;64(22):2994-3006.doi:10.1007 / s00018-007-7345-y)https: / / pubmed.ncbi.nlm.nih.gov / 17938858 / . We considered both CDS1(cHP) and CDS2(SL427) target disruption structures to be SRVVLC. Pirakitikulr et al.2016 (Molecular Cell 62,111-120 April 7,2016) a This was considered to be SRVVLC as defined in 2016 Elsevier Inc. (http: / / dx.doi.org / 10.1016 / j.molcel.2016.01.024).

[0250] Cell culture and transfection The inventors transfected Huh7 cells with each circRNA and infected the cells with an HCV derivative expressing luciferase 24 hours after transfection. 48 hours after infection, the inventors measured luciferase levels and compared them to the control (pseudo-transfected) cells. The inventors performed five biological replicates for each condition. All tested circRNAs significantly inhibited HCV infection. As shown in Figure 7, all designed circRNAs could reduce infection by varying efficiencies ranging from 30% to 70% compared to the control.

[0251] Example 2: The inventors designed and tested three circRNAs (circ_dv_3utr (SEQ ID NO: 8), circ_dv_cHP_v1 (SEQ ID NO: 9), and circ_dv_cHP_v2 (SEQ ID NO: 10)) on two different regions (SEQ ID NO: 7) of the DENV (dengue fever) genome, as previously disclosed in the section on circRNA generation. Plasmids for generating DENV (pFK-DVs-R2A) possessing a ferase reporter gene have been previously described (Scaturro, P. et al. Characterization of the mode of action of a potent dengue virus capsid inhibitor. J. Virol. 88, 11540-55 (2014)). All three circRNAs contain seven hybridization regions targeting corresponding regions of the DENV genome (see Figure 8 and Sequence ID No. 7). Target disruption structures corresponding to circ_dv_3utr(sHP) were described in Huber et al. 2019 (Nature Communications volume 10, Article number: 1408 (2019)), and the target disruption structure cHP was This was considered to be SRVVLC in Fernandez-Sanles et al. 2017 (Front Microbiol. 2017; 8:546, doi: 10.3389 / fmicb. 2017.00546).

[0252] Cell culture and transfection Human fetal kidney cell line HEK293 was maintained in Dulbecco's modified Eagle medium (DMEM, Invitrogen, Carlsbad, California) supplemented with 10% heat-inactivated fetal bovine serum (FBS) and 10% non-essential amino acids. Cells were grown in an incubator at 37°C and 5% CO2. The day before transfection, 1 × 10⁶ cells were used. 5 HEK293 cells were seeded in 24-well plates at a rate of 1 cells / well. Each plasmid containing circRNA or an empty plasmid was transfected with Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions. After overnight incubation, the DMEM medium was removed and the cells were washed with 1×PBS. The cells were inoculated with dengu virus at 37°C for 4 hours. Finally, the virus-containing medium was replaced with fresh medium. 48 hours after infection, luciferase activity was assayed. The cells were washed with 1×PBS, dissolved in 150 μl of Renilla lysis buffer, and frozen. After thawing, the lysate was resuspended by pipetting. 4 μl of the lysate was mixed with 20 μl of Renilla luciferase assay buffer and 1 / 200 of the substrate from the Renilla luciferase assay system (Promega), and immediately measured on a luminometer for 2 seconds. The mean relative light units (RLU) were plotted as a percentage relative to the control infection (cells transfected with the circoVIR plasmid).

[0253] The results are shown in Figure 9, demonstrating a significant inhibition of DENV infection compared to the control (empty plasmid).

[0254] Example 3: The inventors designed and tested five circRNAs (chikv_5utr1 (SEQ ID NO: 12), chikv_5utr2 (SEQ ID NO: 13), chikv_RSE1 (SEQ ID NO: 14), chikv_RSE2 (SEQ ID NO: 15), and chikv_RE (SEQ ID NO: 39) for three different regions of the CHIKV (Chikungunya) genome (SEQ ID NO: 11), as previously disclosed in the section on circRNA generation. The plasmid for creating CHIKV with a Gaussial ciferase reporter gene (courtesy of Dr. Merits of the University of Tartu, Estonia) was La Reunion (directly based on viral sequences isolated from human patients from isolate LR2006_OPY1 (DQ443544)). All five circRNAs contain six hybridization regions targeting corresponding regions of the CHIKV genome (SEQ ID NO: 11). Both the targeted disruption structure and the targeted disruption structure "repetitive sequence structure" in the 5'UTR of CHIKV are based on Hossain Khan et al., 2002 (Journal of General Virology (2002), 83, 3075-3084) considered it to be SRVVLC. The recoding element, which is the third target disruption structure of CHIKV, was considered to be SRVVLC in A. Kendra et al., 2018 (J. Biol. Chem. (2018) 293(45) 17536-17545).

[0255] Cell culture and transfection Human fetal kidney cell line HEK293 was maintained in Dulbecco's modified Eagle medium (DMEM, Invitrogen, Carlsbad, California) supplemented with 10% heat-inactivated fetal bovine serum (FBS) and 10% non-essential amino acids. Cells were grown in an incubator at 37°C and 5% CO2. The day before transfection, 1 × 10⁶ cells were used. 5HEK293 cells were seeded in 24-well plates at a rate of 1 / well. Each plasmid containing circRNA or an empty plasmid was transfected with Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions. After overnight incubation, the DMEM medium was removed and the cells were washed with 1×PBS. The cells were inoculated with chikungunya virus at 37°C for 1 hour. Finally, the virus-containing medium was replaced with fresh medium. Luciferase activity was assayed 16 hours after infection. Cell supernatant was collected and inactivated with UV for 10 minutes. 4 μl of supernatant was mixed with 20 μl of Renilla luciferase assay buffer and 1 / 200 of the substrate from the Renilla luciferase assay system (Promega), and immediately measured on a luminometer for 2 seconds. Mean relative luminal units (RLU) were plotted as a percentage relative to control infection (cells transfected with circoVIR plasmid).

[0256] The results are shown in Figure 10, demonstrating significant inhibition of CHIKV infection compared to the control (empty plasmid).

[0257] Example 4: The inventors designed and tested three circRNAs (DENV1 cHP_HCV CDS2_1 (SEQ ID NO: 17), DENV cHP_HCV CDS2_2 (circ_dv_cHP_v1_circ_hcv_cds2_2, SEQ ID NO: 32), and (DENV1 cHP_HCV CDS2_T (SEQ ID NO: 16)) on one region of the DENV (dengue fever) genome (SEQ ID NO: 7) and one region of the HCV genome (SEQ ID NO: 1), as previously disclosed in the section on circRNA generation. Plasmids for producing HCV (pFK-Luc-Jc1) containing a firefly luciferase reporter gene have been previously described (Wakita T et al., Production of infectious hepatitis C virus in tissue culture from a cloned viral genome, Nat). Med, 2005;11:791-796). A plasmid for producing DENV (pFK-DVs-R2A) possessing the sea urticaria luciferase reporter gene has been previously described (Scaturro, P. et al., Characterization of the mode of action of a potent dengue virus capsid inhibitor, J. Virol. 88, 11540-55 (2014)).

[0258] The first two circRNAs contain six hybridization regions targeting corresponding regions in the DENV and HCV genomes, while the last one contains seven hybridization regions for DENV and twelve for HCV.

[0259] Cell culture and transfection Human fetal kidney cell line HEK293 was maintained in Dulbecco's modified Eagle medium (DMEM, Invitrogen, Carlsbad, California) supplemented with 10% heat-inactivated fetal bovine serum (FBS) and 10% non-essential amino acids. Cells were maintained at 37°C and 5% CO2. They were grown in an incubator. The day before transfection, 1 × 10 5HEK293 cells were seeded in 24-well plates at a rate of 1 cells / well. Each plasmid containing circRNA or an empty plasmid was transfected with Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions. After overnight incubation, the DMEM medium was removed and the cells were washed with 1×PBS. The cells were inoculated with dengu virus at 37°C for 4 hours. Finally, the virus-containing medium was replaced with fresh medium. 48 hours after infection, luciferase activity was assayed. The cells were washed with 1×PBS, dissolved in 150 μl of Renilla lysis buffer, and frozen. After thawing, the lysate was resuspended by pipetting. 4 μl of the lysate was mixed with 20 μl of Renilla luciferase assay buffer and 1 / 200 of the substrate from the Renilla luciferase assay system (Promega), and immediately measured on a luminometer for 2 seconds. The mean relative light units (RLU) were plotted as a percentage relative to the control infection (cells transfected with the circoVIR plasmid).

[0260] Human hepatocellular carcinoma cell line Huh7 / Scr was maintained in Dulbecco's modified Eagle medium (DMEM, Invitrogen, Carlsbad, California) supplemented with 10% thermo-inactivated fetal bovine serum (FBS) and 10% non-essential amino acids. Cells were grown in an incubator at 37°C and 5% CO2. 2.5 × 10⁶ cells were measured the day before transfection. 4 Human Huh7 / Scr cells were seeded in 24-well plates. Each plasmid containing circRNA or an empty plasmid was transfected with Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions.

[0261] After overnight incubation, the DMEM medium was removed and the cells were washed with 1×PBS. Various viruses were inoculated into the cells at 37°C for 4 hours. Finally, the virus-containing medium was replaced with fresh medium. 48 hours after infection, luciferase activity was assayed. The cells were washed with 1×PBS, dissolved in 150 μl of Passive lysis buffer, and frozen. After thawing, the lysate was resuspended by pipetting. 50 μl of the lysate was mixed with 25 μl of luciferase assay reagent (Promega) and incubated at room temperature for 5 minutes. Luciferase activity was then measured using a luminometer for 2 seconds. Mean relative luminescence units (RLU) were plotted as a percentage relative to control infection (cells transfected with circoVIR plasmid).

[0262] The results for each circRNA against both DENV and HCV are shown in Figures 11, 12, 13, 14, and 24, along with the corresponding positive and negative controls from Examples 1 and 2. Inhibition was shown for all circRNAs in both viral infections, demonstrating the broad spectral capability of the designed circRNAs.

[0263] Example 5: The inventors designed and tested two circRNAs (circ_wnv_slII_1 (SEQ ID NO: 24) and circ_wnv_slII_2 (SEQ ID NO: 19)) against a single region of the WNV (West Nile virus) genome (SEQ ID NO: 20), as previously disclosed in the section on circRNA generation. Plasmids for generating WNV with the Nanoluc luciferase reporter gene were kindly provided by Dr. Merits of the University of Tartu, Estonia. Both circRNAs contain seven hybridization regions targeting the corresponding region of the WNV genome. The targeted disruption structure SLII was considered to be SRVVLC in Fernandez-Sanles et al., 2017 (Front Microbiol. 2017; 8:546 doi: 10.3389 / fmicb. 2017.00546).

[0264] Cell culture and transfection Human fetal kidney cell line HEK293 was maintained in Dulbecco's modified Eagle medium (DMEM, Invitrogen, Carlsbad, California) supplemented with 10% heat-inactivated fetal bovine serum (FBS) and 10% non-essential amino acids. Cells were grown in an incubator at 37°C and 5% CO2. The day before transfection, 1 × 10⁶ cells were used. 5 HEK293 cells were seeded at a rate of 1 / well in 24-well plates. Each plasmid containing circRNA or an empty plasmid was transfected with Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions. After overnight incubation, the DMEM medium was removed and the cells were washed with 1×PBS. The cells were inoculated with WNV at 37°C for 4 hours. Finally, the virus-containing medium was replaced with fresh medium. 48 hours after infection, luciferase activity was assayed. The cells were washed with 1×PBS, dissolved in 150 μl of Renilla lysis buffer, and frozen. After thawing, the lysate was resuspended by pipetting. 4 μl of the lysate was mixed with 20 μl of Renilla luciferase assay buffer and 1 / 200 of the substrate from the Renilla luciferase assay system (Promega), and immediately measured on a luminometer for 2 seconds. The mean relative light units (RLU) were plotted as a percentage relative to the control infection (cells transfected with the circoVIR plasmid).

[0265] The results are shown in Figure 15, demonstrating a significant inhibition of WNV infection compared to the control (empty plasmid).

[0266] Example 6: The inventors designed and tested three circRNAs (dchp_wsIlI_A (SEQ ID NO: 21), dchp_wsIlI_B (SEQ ID NO: 22), and dchp_wsIlI_C (SEQ ID NO: 23) for one region of the DENV (dengue fever) genome (SEQ ID NO: 7) and one region of the WNV genome (SEQ ID NO: 20), as previously disclosed in the section on circRNA generation. Plasmids for producing DENV (pFK-DVs-R2A) possessing a sea urticaria luciferase reporter gene have been previously described (Scaturro, P. et al., Characterization of the mode of action of a potent dengue virus capsid). inhibitor, J.Virol.88,11540-55(2014). The plasmid for constructing WNVs containing the Nanoluc luciferase reporter gene was generously provided by Dr. Merits of the University of Tartu, Estonia. The circRNA contains six hybridization regions that target corresponding regions of the DENV and WNV genomes.

[0267] Cell culture and transfection Human fetal kidney cell line HEK293 was maintained in Dulbecco's modified Eagle medium (DMEM, Invitrogen, Carlsbad, California) supplemented with 10% heat-inactivated fetal bovine serum (FBS) and 10% non-essential amino acids. Cells were grown in an incubator at 37°C and 5% CO2. The day before transfection, 1 × 10⁶ cells were used. 5HEK293 cells were seeded in 24-well plates at a rate of 1 / well. Each plasmid containing circRNA or an empty plasmid was transfected with Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions. After overnight incubation, the DMEM medium was removed and the cells were washed with 1×PBS. The cells were inoculated with dengue virus or West Nile virus at 37°C for 4 hours. Finally, the virus-containing medium was replaced with fresh medium. 48 hours after infection, luciferase activity was assayed. The cells were washed with 1×PBS, dissolved in 150 μl of Renilla lysis buffer, and frozen. After thawing, the lysate was resuspended by pipetting. 4 μl of the lysate was mixed with 20 μl of Renilla luciferase assay buffer and Renilla luciferase The substrate was mixed with 1 / 200 of the ze assay system (Promega) and immediately measured with a luminometer for 2 seconds. The mean relative luminal units (RLU) were plotted as a percentage relative to the control infection (cells transfected with circoVIR plasmid).

[0268] The results for each circRNA against both DENV and WNV are shown in Figures 16 and 17, along with the corresponding positive and negative controls from Examples 2 and 5. Inhibition of circRNAs in both viral infections is demonstrated, showing the broad spectral capability of the designed circRNAs.

[0269] Example 7: The inventors tested whether circ_hcv_cds2 (SEQ ID NO: 6) could inhibit already established infections. To this end, Huh7 / Scr cells were infected with HCVJc1-luc and transfected with circ_hcv_cds2 (SEQ ID NO: 6) 48 hours later. Luciferase levels were measured 24 hours after transfection. Importantly, circ_hcv_cds2 inhibited infectivity with the same efficiency as when the cells expressed circ_hcv_cds2 before infection (Figure 18).

[0270] Example 8: Next, the inventors investigated whether circ_hcv_cds2 (SEQ ID NO: 6) actually inhibits the functions described for target sequence and viral RNA replication. For this purpose, the inventors used HCV RNA replicons that have a luciferase reporter gene but lack the viral structural gene necessary for capsid formation. Therefore, these replicons enable efficient translation and replication of the viral RNA genome, but do not enable viral particle generation. Huh7 / Scr cells were transfected with circ_hcv_cds2 (SEQ ID NO: 6) or the corresponding empty plasmid, and the following day, they were transfected with the HCV replicons. Luciferase levels were measured 4 hours and 48 hours after HCV replicon transfection. These times were chosen because established dynamics have shown that luciferase production at 4 hours is solely due to HCV RNA translation, while at 48 hours it is due to both translation and replication. In fact, a decrease in viral infectivity was observed at 48 hours post-transfection, but not at 4 hours (Figure 19), indicating that circ_hcv_cds2 impairs viral RNA replication.

[0271] Example 9: Circ_dv_3utr (SEQ ID NO: 8) and circ_dv_cHP_v1 (SEQ ID NO: 9), designed to target structures within the DENV RNA genome that direct RNA replication, were tested to determine whether they inhibit DENV RNA replication following the same procedure as in Example 8. The results showed that circ_dv_3utr and circ_dv_cHP_v1 inhibited luciferase expression levels at 48 hours when the RNA genome was translated and replicated, but did not inhibit it at 8 hours when translated alone (Figure 20).

[0272] Example 10: The human embryonic kidney cell line HEK293 and the hepatocellular carcinoma cell line Huh7 were maintained in Dulbecco's Modified Eagle Medium (DMEM, Invitrogen, Carlsbad, CA) supplemented with 10% heat-inactivated fetal bovine serum (FBS) and 10% non-essential amino acids. The cells were grown in an incubator at 37°C and 5% CO2. The day before transfection, 1×10 5 HEK293 cells / well or 4×10 4 Huh7 cells / well were seeded in 24-well plates. According to the manufacturer's instructions, Lipofectamine 2000 (Invitrogen) was used to transfect 100 nanograms of circular RNA (circWD1 - in vitro SEQ ID NO: 63) against WNV - DENV or circular RNA (circHCV - in vitro SEQ ID NO: 64) against HCV . After an overnight incubation, the DMEM medium was removed and the cells were washed with 1×PBS. HEK293 cells were inoculated with dengue virus or West Nile virus at 37°C for 4 hours. In parallel, Huh7 cells were inoculated with hepatitis C virus at 37°C for 4 hours. Finally, the virus-containing medium was replaced with fresh medium. 48 hours after infection, luciferase activity was assayed. The cells were washed with 1×PBS, lysed in 150 μl of Renilla lysis buffer (for HEK293) or 150 μl of Passive lysis buffer, and frozen. After thawing, the lysates were resuspended by pipetting. For DENV and WNV, 4 μl of the lysate was mixed with 20 μl of Renila luciferase assay buffer and 1 / 200 substrate from the Renilla luciferase assay system (Promega) and immediately measured in a luminometer for 2 seconds. For HCV, 50 μl of the lysate was mixed with 25 μl of luciferase assay reagent (LARII) for 5 minutes and immediately measured in a luminometer for 2 seconds. The mean relative light units (RLU) were plotted in Figure 26 as a percentage relative to mock-infected cells (cells transfected with the circoVIR plasmid).

[0273] Example 11: The inventors have identified 12 circRNAs (circ_SARS_tA_4 (SEQ ID NO: 49), circ_SARS_tA_5 (SEQ ID NO: 68), circ_SARS_tA_6 (SEQ ID NO: 69), circ_SARS_tA_7 (SEQ ID NO: 70), circ_SARS_tC_3 (SEQ ID NO: 52), circ_SARS_tC_5 (SEQ ID NO: 71), circ_SARS_tC_6 (SEQ ID NO: 72), circ_SARS_3utr_6 (SEQ ID NO: 63), circ_SARS_3utr_7 (SEQ ID NO: 89), ci rc_SARS_3utr_8 (SEQ ID NO: 65), circ_SARS_5utr_4 (SEQ ID NO: 66), and circ_SARS_5utr_5 (SEQ ID NO: 67) were designed and tested against four regions of the SARS-CoV-2 genome (SEQ ID NO: 58; SEQ ID NO: 59; SEQ ID NO: 60; SEQ ID NO: 62), as previously disclosed in the section on circRNA generation. The SARS-CoV-2 strain hCoV-19 / Spain / VH000001133 / 2020 (EPI_ISL_418860) was kindly provided by Miguel Chillon of the Autonomous University of Barcelona. The designed circRNAs contain 6-7 hybridization regions targeting the corresponding regions in the SARS-CoV-2 genome. The replication sites of the target disruption structures were identified in J. Goebel et al., 2004 (American Society). The SRVVLC was considered to be the same as in *for Microbiology.Journal of Virology Volume 78, Issue 14, Pages 7846-7851 https: / / doi.org / 10.1128 / JVI.78.14.7846-7851.2004*. The target disruption structure SL-2 was considered to be the SRVVLC in Chen et al., 2010 (Virology 401(2010)29-41 doi:10.1016 / j.virol.2010.02.007). The remaining target disruption structures (A, C, and D) were considered to be the virus-conserved structures in Rangan et al., 2020 (doi:https: / / doi.org / 10.1101 / 2020.03.27.012906) according to the RNAz approach described above.

[0274] Example 12: Cell culture and transfection Green monkey kidney epithelial cell line VERO E6 was maintained in Dulbecco's modified Eagle medium (DMEM, Invitrogen, Carlsbad, California) supplemented with 10% heat-inactivated fetal bovine serum (FBS) and 10% non-essential amino acids. Cells were grown in an incubator at 37°C and 5% CO2. On the day before transfection, 6 × 10⁶ cells were placed per well. 4 100 VERO E6 cells were seeded in a 24-well plate. Each plasmid containing circRNA or an empty plasmid was transfected with Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions. After overnight incubation, the DMEM medium was removed and the cells were washed with 1×PBS. The cells were inoculated with SARS-CoV-2 at 37°C for 1 hour. Finally, the cells were transfected. The SARS-CoV-2 culture medium was replaced with fresh medium. 48 hours after infection, the supernatant was collected, inactivated, and viral RNA was extracted using the Quick-RNA Viral Kit (Zymo Research, Irvine, USA). SARS-CoV-2 RNA levels were measured by qPCR using the qScript XLT One-Step RT-qPCR ToughMix, ROX (Quanta Biosciences, Beverly, USA) with the specific probe 2019 nCoV_N1-P, 5'-FAM-ACCCCGCATTACGTTTGGTGGACC-BHQ 1-3' (SEQ ID NO: 73) and the primers 2019-nCoV_N1-F, 5'-GACCCCAAAATCAGCGAAAT-3' (SEQ ID NO: 74) and 2019-nCoV_N1-R, 5'-TCTGGTTACTGCCAGTTGAATCTG-3' (SEQ ID NO: 75) (Biomers, Ulm, Germany). The mean relative RNA levels were plotted as a percentage relative to the control infection (cells transfected with the circoVIR plasmid).

[0275] The results for each circRNA against SARS-CoV-2 are shown in Figure 33. Inhibition was demonstrated for circRNAs in SARS-CoV-2 virus infection, demonstrating the antiviral effect of the designed circRNAs.

[0276] Example 13: Green monkey kidney epithelial cell line VERO E6 was maintained in Dulbecco's modified Eagle medium (DMEM, Invitrogen, Carlsbad, California) supplemented with 10% heat-inactivated fetal bovine serum (FBS) and 10% non-essential amino acids. Cells were grown in an incubator at 37°C and 5% CO2. On the day before transfection, 6 × 10⁶ cells were placed per well. 4100 nanograms of VERO E6 cells were seeded in 24-well plates. Following the manufacturer's instructions, 100 nanograms of circular RNA for SARS-CoV-2 (circSARS-in vitro SEQ ID NO: 70) or circular RNA for WNV-DENV (circWD1-in vitro SEQ ID NO: 21) were transfected using lipofectamine 2000 (Invitrogen). After overnight incubation, the DMEM medium was removed and the cells were washed with 1×PBS. SARS-CoV-2 was inoculated into the cells at 37°C for 1 hour. Finally, the virus-containing medium was replaced with fresh medium. 48 hours after infection, the supernatant was collected, inactivated, and viral RNA was extracted using the Quick-RNA Viral Kit (Zymo Research, Irvine, USA). SARS-CoV-2 RNA levels were measured by qPCR using the qScript XLT One-Step RT-qPCR ToughMix, ROX (Quanta Biosciences, Beverly, USA) with the specific probe 2019 nCoV_N1-P, 5'-FAM-ACCCCGCATTACGTTTGGTGGACC-BHQ 1-3' (SEQ ID NO: 73) and primers 2019-nCoV_N1-F, 5'-GACCCCAAAATCAGCGAAAT-3' (SEQ ID NO: 74) and 2019-nCoV_N1-R, 5'-TCTGGTTACTGCCAGTTGAATCTG-3' (SEQ ID NO: 75) (Biomers, Ulm, Germany). The mean relative RNA levels were plotted as a percentage relative to control infection (cells transfected with unrelated circRNA) in Figure 34.

[0277] Example 14: In this embodiment, we present several RNAiFold input files to check whether the target disruption structure can actually be disrupted by hybridization (from there, the design of the circRNA and the hybridization region that disrupts it follows immediately).

[0278] Regarding the HCV target destruction structure cHP and the corresponding target hybridization region (SEQ ID NO: 27, highlighted in bold): TIFF2026102653000026.tif26170

[0279] The input "((((((((((((((((((((((((((&)))))))))))))))))))))))))),,,,,,,,,,,,,,,,,,,,,,,,,,," means that the software should look for a nucleotide sequence that is the knucleotide length, and in the minimum free energy structure, the nucleotide located at the position corresponding to the open parenthesis "(" will be the base paired with the nucleotide located at the position corresponding to the closed parenthesis ")". The comma "," means that the nucleotide at the corresponding position can form a base pair or cannot exist in the minimum free energy structure. The & symbol separates both strands. Note that the base-paired nucleotides on the second strand (target disruption structure) correspond to the target hybridization region.

[0280] Furthermore, input: TIFF2026102653000027.tif12170 means that the nucleotides available for selection are fixed, where N represents any nucleotide (A, C, G, or U). In this case, the nucleotides corresponding to the target disruption structure are fixed, while all nucleotides corresponding to the hybridization region are not fixed (N - any nucleotide). The results are shown below: TIFF2026102653000028.tif103170

[0281] Regarding the CHIKV target destruction structure RSE and the corresponding target hybridization region (SEQ ID NO: 35): TIFF2026102653000029.tif39170

[0282] The results are shown below: UUGCGUGGCCCUUUGGUCUGUAGGUUGUUUGCU&AGCAAAUAAUCUAUAGAUCAAAGGGCUACGCAACCCCUGAA UUGUGUGGCCCUUUGGUCUGUAGGUUGUUUGCU&AGCAAAUAAUCUAUAGAUCAAAGGGCUACGCAACCCCUGAA UUGCGUAGCCCUUUGGUCUGUAGGUUGUUUGCU&AGCAAAUAAUCUAUAGAUCAAAGGGCUACGCAACCCCUGAA UUGUGUAGCCCUUUGGUCUGUAGGUUGUUUGCU&AGCAAAUAAUCUAUAGAUCAAAGGGCUACGCAACCCCUGAA UUGCGUGGUCCUUUGGUCUGUAGGUUGUUUGCU&AGCAAAUAAUCUAUAGAUCAAAGGGCUACGCAACCCCUGAA UUGUGUGGUCCUUUGGUCUGUAGGUUGUUUGCU&AGCAAAUAAUCUAUAGAUCAAAGGGCUACGCAACCCCUGAA UUGCGUAGUCCUUUGGUCUGUAGGUUGUUUGCU&AGCAAAUAAUCUAUAGAUCAAAGGGCUACGCAACCCCUGAA UUGUGUAGUCCUUUGGUCUGUAGGUUGUUUGCU&AGCAAAUAAUCUAUAGAUCAAAGGGCUACGCAACCCCUGAA UUGCGUAGCUCUUUGGUCUGUAGGUUGUUUGCU&AGCAAAUAAUCUAUAGAUCAAAGGGCUACGCAACCCCUGAA UUGUGUAGCUCUUUGGUCUGUAGGUUGUUUGCU&AGCAAAUAAUCUAUAGAUCAAAGGGCUACGCAACCCCUGAA

[0283] Regarding the WNV target destruction structure SLII and the corresponding target hybridization region (SEQ ID NO: 37): TIFF2026102653000030.tif39170

[0284] The results are shown below: TIFF2026102653000031.tif103170

[0285] Regarding the SAR-Cov2 target destruction structure replication site and the corresponding target hybridization region (SEQ ID NO: 62): TIFF2026102653000032.tif39170

[0286] The results are shown below: TIFF2026102653000033.tif134170

[0287] In all cases, including those not shown above, RNAiFold returns multiple solutions, namely sequences corresponding to hybridization regions that may disrupt the corresponding target disruption structure through hybridization.

[0288] Finally, the inventors present herein a well-established dot-bracket notation used in the present invention to obtain the target hybridization region (highlighted in bold herein) for each virus: TIFF2026102653000034.tif5170 Target 1: IRES1 (Domain II) Sequence ID 76, Figure 1a from Romero et al. 2007 (Cell Mol Life Sci. 2007 Nov;64(22):2994-3006.doi:10.1007 / s00018-007-7345-y.): TIFF2026102653000035.tif20170 Target 2: IRES 2 (Domain IV) Sequence ID 77, Figure 1a from Romero et al. (Cell Mol Life Sci. 2007 Nov;64(22):2994-3006.doi:10.1007 / s00018-007-7345-y.): TIFF2026102653000036.tif12170 target 3: CDS 1(cHP) SEQ ID NO: 78; Figure S1 B of Pirakitikulr et al. 2016 (Pirakitikulr et al., 2016, Molecular Cell 62, 111-120 April 7, 2016 Elsevier Inc.) TIFF2026102653000037.tif11170 Target 4: CDS2(SL427) SEQ ID NO: 79; Figure 2a of Pirakitikulr et al. 2016 (Pirakitikulr et al., 2016, Molecular Cell 62, 111-120 April 7, 2016 Elsevier Inc). TIFF2026102653000038.tif22170TIFF2026102653000039.tif5170 Target 1:sHP Sequence ID 29; Figure 1c of Huber et al., 2019 (Huber et al. Nature Communications volume 10, Article number: 1408, 2019). TIFF2026102653000040.tif10170 target 2:cHP Sequence ID 30; Figure 2B of Sanles et al. 2017 (Sanles et al. 2017. Front Microbiol.; 8:546.): TIFF2026102653000041.tif11170TIFF2026102653000042.tif5170 target 1:5'UTR: Sequence ID 80, Figure 3A from Khan et al. 2002 (Khan et al. Journal of General Virology Volume 83, Issue 12): TIFF2026102653000043.tif22170 Target 2: Iterative Array Elements: Sequence ID 81, Figure 4B of Khan et al. 2002 (Khan et al. Journal of General Virology Volume 83, Issue 12): TIFF2026102653000044.tif12170 Target 3: Re-coding element: Sequence ID 82, Kendra et al. 2018 (Kendra et al. 2 Figure 4D from 018 Protein Synthesis and Degradation (Volume 293, Issue 45, pp. 17536-17545, November 09, 2018): TIFF2026102653000045.tif22170TIFF2026102653000046.tif6170 Target 1:SLII: Sequence ID 83; Figure 3 of Sanles et al. 2017 (Sanles et al. 2017. Front Microbiol.; 8:546.): TIFF2026102653000047.tif11170TIFF2026102653000048.tif6170 Target 1: Completely conserved replication sites in pangolins and RatG13 Sequence ID No. 84, Figure 1 from Goebel et al. 2004 (Goebel et al., Journal of Virology, July 2004, pp. 7846-7851): TIFF2026102653000049.tif10170 Target 2:SL-2, Sequence ID 58, Cheng Chen et al. 2010 (Cheng Chen Figure 3b (et al. Virology, Volume 401, Issue 1, 25 May 2010, Pages 29-41) TIFF2026102653000050.tif10170 Target 3: Target A Sequence ID 85, Figure 2 of Rangan et al. 2020 (Rangan et al. bioRxiv preprint doi:https: / / doi.org / 10.1101 / 2020.03.27.012906): TIFF2026102653000051.tif11170 Target 4: Target C Sequence ID 86, Figure 2 of Rangan et al. 2020 (Rangan et al. bioRxiv preprint doi:https: / / doi.org / 10.1101 / 2020.03.27.012906): TIFF2026102653000052.tif12170 Target 5: Target D Sequence ID 87, Figure 2 of Rangan et al. 2020 (Rangan et al. bioRxiv preprint doi:https: / / doi.org / 10.1101 / 2020.03.27.012906): TIFF2026102653000053.tif11170

[0289] Further items of the present invention This invention also includes the following items:

[0290] 1. An artificial circular RNA of 200-600 nucleotides having 6-20 hybridization regions between 10-50 nucleotides in size relative to one or more structural regions of one or more RNA viral genomes, wherein such hybridization regions have sequences distinct from each other, and one or more structural regions are structural regions essential to the viral life cycle (SRVVLC) preceded by single-stranded regions, and the artificial circular RNA can disrupt the structure of one or more SRVVLC, thereby reducing the infectivity of the virus.

[0291] 2. The hybridization region is a) Separated by non-hybridization regions up to 20 nucleotides in size, or b) Not separated by non-hybridization regions, or c) Duplicate, Artificial circular RNA as described in item 1.

[0292] 3. An artificial circular RNA according to item 1 or 2, wherein the target of the hybridization region is the SRVVLC of the IRES element of the viral genome.

[0293] 4. An artificial circular RNA as described in item 1 or 2, wherein the target of the hybridization region is the SRVVLC of the 5'UTR of the viral genome.

[0294] 5. An artificial circular RNA as described in item 1 or 2, wherein the target of the hybridization region is the SRVVLC of the CDS of the viral genome.

[0295] 6. An artificial circular RNA as described in item 1 or 2, wherein the target of the hybridization region is the SRVVLC of the 3'UTR of the viral genome.

[0296] 7. An artificial circular RNA according to any one of items 1 to 6, wherein the target of the hybridization region is a combination of two or more IRES elements, 5'UTR regions, CDS regions, or 3'UTR regions of the viral genome.

[0297] 8. An artificial circular RNA as described in item 3 or 7, wherein the viral genome is selected from HCV, HAV, poliovirus, coxsackie B virus, and rhinovirus (common cold).

[0298] 9. An artificial circular RNA as described in any of items 4-7, wherein the viral genome is selected from HCV, dengue fever, Zika, chikungunya, West Nile, and yellow fever viruses.

[0299] 10. An artificial circular RNA according to any of items 1 to 9, wherein the circular RNA has broad spectral activity against two or more RNA virus genomes.

[0300] 11. A composition comprising the artificial circular RNA described in any of the above items.

[0301] 12. A kit comprising an artificial circular RNA described in any of items 1 to 10 or a composition described in item 11, and instructions for using the said circular RNA or composition.

[0302] 13. The aforementioned circular RNA is sequence numbers 2, 4, 5, 6 (in the case of HCV); sequence numbers 8, 9 One or more targets of the hybridization region comprising 6 to 20 sequences, including sequences selected from: 10 (in the case of dengue virus); SEQ ID NOs: 12, 13, 14, 15, 39 (in the case of chikungunya virus); SEQ ID NOs: 16 and 17 (broad-spectrum activity against both HCV and dengue virus); SEQ ID NOs: 24 and 19 (in the case of West Nile virus); SEQ ID NOs: 21, 22 and 23 (broad-spectrum activity against both dengue and West Nile virus); SEQ ID NOs: 32 (broad-spectrum activity against both HCV and dengue virus). An artificial circular RNA as described in any of items 1 to 10, contained in SEQ ID NOs: 1, 7, 11 and / or 20, or a composition as described in item 11, or a kit as described in item 12.

[0303] 14. Artificial circular RNA as described in any of items 1-10 or 13, or a composition as described in item 11 or 13, or a kit as described in item 12 or 13, for use in the prevention and / or treatment of viral infection.

[0304] 15. Artificial circular RNA or composition or kit for use as described in item 14, wherein the viral infection is caused by HCV, HAV, poliovirus, coxsackie B virus, rhinovirus (common cold), dengue fever, Zika, chikungunya, West Nile, or yellow fever virus.

Claims

1. An artificial circular RNA suitable for disrupting one or more target disruption structures of one or more RNA fragments by hybridization, (a) The artificial circular RNA comprises 150 to 800 nucleotides, preferably 200 to 600 nucleotides, (b) The artificial circular RNA comprises two or more hybridization regions, (i) Completely hybridize with at least one target hybridization region included in the one or more target disruption structures of the one or more RNA fragments, (ii) Having a total of 7 to 100 nucleotides, preferably 10 to 50 nucleotides, (c) The one or more target destruction structures are (i) The region of the unpaired nucleotide includes at least a hairpin loop before or after it, (ii) comprising at least one target hybridization region comprising at least 2 nucleotides, preferably 3 nucleotides or more, single-stranded regions before or after a double-stranded region of at least 5 nucleotides, preferably 10 nucleotides or more, wherein the at least one target hybridization region completely hybridizes with each of the two or more hybridization regions of the artificial circular RNA. (d) An artificial circular RNA further characterized by the fact that when the two or more hybridization regions contained in the artificial circular RNA hybridize with the target hybridization region, the hybridization energy measured by RNAcofold between the hybridization region and the at least one target hybridization region is negative compared to the energy of the target disruption region, thereby disrupting the target disruption structure.

2. The artificial circular RNA according to claim 1, wherein the artificial circular RNA comprises 6 to 20 hybridization regions.

3. The artificial circular RNA according to claim 2, wherein at least two, and preferably all, of the hybridization regions can completely hybridize with the same target hybridization region.

4. The artificial circular RNA according to any one of claims 2 to 3, wherein at least two, preferably all, of the hybridization regions have different nucleotide sequences.

5. The two or more hybridization regions described above a) Separated by non-hybridization regions up to 20 nucleotides in size; or b) Not separated by non-hybridization regions; or c) Duplicate, The artificial circular RNA according to any one of claims 2 to 4.

6. The artificial circular RNA according to any one of claims 1 to 5, wherein one or more RNA fragments are selected from mRNA, tRNA, rRNA, non-coding RNA, and viral genomic RNA.

7. The artificial circular RNA according to claim 6, wherein one or more RNA fragments are viral genomic RNA.

8. The artificial circular RNA according to claim 7, wherein one or more RNA fragments are positive sense single-stranded viral genome RNAs.

9. The artificial circular RNA according to any one of claims 6 to 7, wherein the viral genome RNA is selected from influenza virus, HAV, poliovirus, coxsackie B virus, coronavirus and rhinovirus (common cold).

10. The artificial circular RNA according to any one of claims 6 to 8, wherein the viral genome RNA is selected from hepatitis C virus, dengue fever, Zika, Chikungunya, West Nile virus, and yellow fever virus.

11. The artificial circular RNA according to any one of claims 6 to 8, wherein the viral genomic RNA is derived from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).

12. The artificial circular RNA according to any one of claims 7 to 11, wherein at least one of the target disruption structures is selected from the group consisting of the following: (a) Internal ribosome entry (IRES) domains IV and V, capsid coding region hairpin element (cHP), or SL427 derived from hepatitis C virus, (b) Short stem-loop (sHP) or capsid-code region hairpin element (cHP) derived from dengue virus, (c) 5' untranslated regions (5'UTRs), repeating sequence elements (RSEs), or recoding elements derived from Chikungunya, (d) Stem loop III (SLIII) originating from the West Nile, and / or (e) SL-2, replication site, target A, target C, target D derived from coronavirus, preferably severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).

13. The artificial circular RNA according to any one of claims 7 to 12, wherein the at least one of the target disruption structures includes a target disruption structure selected from the group consisting of SEQ ID NOs: 76, 77, 78 and / or 79 derived from hepatitis C virus, and the target hybridization region is selected for each of these target disruption structures from SEQ ID NOs: 25, 26, 27 and 28, respectively.

14. The artificial circular RNA according to any one of claims 7 to 12, wherein the at least one of the target disruption structures includes a target disruption structure selected from the group consisting of SEQ ID NOs. 29 and / or 30 derived from dengue virus, and the target hybridization region is selected from SEQ ID NOs. 29 and 30 for each of these target disruption structures, respectively.

15. The artificial circular RNA according to any one of claims 7 to 12, wherein the at least one of the target disruption structures includes a target disruption structure selected from the group consisting of SEQ ID NOs: 80, 81 and / or 82 derived from Chikungunya virus, and the target hybridization region is selected for each of these target disruption structures from SEQ ID NOs: 33, 35 and 31, respectively.

16. The artificial circular RNA according to any one of claims 7 to 12, wherein the at least one of the target disruption structures includes the target disruption structure of Sequence ID No. 83 derived from the West Nile, and the target hybridization region is Sequence ID No.

37.

17. The artificial circular RNA according to any one of claims 7 to 12, wherein the at least one of the target disruption structures includes a target disruption structure selected from the group consisting of SEQ ID NOs: 84, 58, 85, 86 and / or 87 derived from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), and the target hybridization region is selected for each of these target disruption structures from SEQ ID NOs: 62, 58, 59, 60 and 61, respectively.

18. The artificial circular RNA according to any one of claims 7 to 12, wherein the at least one of the target disruption structures includes a target disruption structure selected from the group consisting of SEQ ID NOs: 30 and / or 79 derived from dengue virus and hepatitis C virus, and the target hybridization region is selected from SEQ ID NOs: 30 and 28 for each of these target disruption structures.

19. The artificial circular RNA according to any one of claims 7 to 12, wherein the at least one of the target disruption structures includes a target disruption structure selected from the group consisting of SEQ ID NOs: 30 and / or 83 derived from dengue virus and West Nile virus, and the target hybridization region is selected from SEQ ID NOs: 30 and 37 for each of these target disruption structures, respectively.

20. A composition comprising artificial circular RNA as defined in any one of claims 1 to 19.

21. A kit comprising an artificial circular RNA as defined in any one of claims 1 to 19 or a composition as defined in claim 20, and instructions for using the artificial circular RNA or composition.

22. The sequences of the artificial RNA are: SEQ ID NOs: 2, 3, 4, 5, 6 (for hepatitis C virus); SEQ ID NOs: 8, 9, 10 (for dengue virus); SEQ ID NOs: 12, 13, 14, 15, 39 (for chikungunya virus); SEQ ID NOs: 16 and 17 (broad-spectrum activity against both hepatitis C virus and dengue virus); SEQ ID NOs: 24 and 19 (for West Nile virus); SEQ ID NOs: 21, 22, and 23 (broad-spectrum activity against both dengue virus and West Nile virus); SEQ ID NOs: 32 (for hepatitis C virus) An artificial circular RNA according to any one of claims 1 to 19, or the composition according to claim 20, or the kit according to claim 21, comprising, or preferably comprising, nucleotides defined in SEQ ID NOs: 36, 38, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 65, 66, 67, 68, 69, 70, 71, 72 (in the case of severe acute respiratory syndrome coronavirus 2) (broad-spectrum activity against both viruses and dengue viruses);

23. The artificial circular RNA according to any one of claims 1 to 19, or the composition according to claim 20, or the kit according to claim 21, wherein one or more target hybridization regions of the artificial circular RNA that completely hybridize with the two or more hybridization regions are included in the artificial RNA defined by SEQ ID NO: 1, SEQ ID NO: 7, SEQ ID NO: 11, SEQ ID NO: 34 and / or SEQ ID NO:

20.

24. An artificial circular RNA according to any one of claims 1 to 19 and 22 and 23, or a composition according to any one of claims 20 to 23, or a kit according to any one of claims 21 to 23, used as a pharmaceutical.

25. An artificial circular RNA according to any one of claims 1 to 19 and 22 to 24, or a composition according to any one of claims 20 to 24, or a kit according to any one of claims 21 to 24, used for a method of preventing and / or treating a viral infection.

26. The aforementioned viral infection is caused by hepatitis C virus, hepatitis A virus, poliovirus, influenza virus, coxsackie B virus, rhinovirus (common cold), dengue fever, zika, chikungunya, West Nile virus, yellow fever virus, or coronavirus, such as SARS and / or MERS, preferably SARS-CoV-2, according to claim 25, the artificial circular RNA according to any one of claims 1 to 19 and 22 to 25, or the composition according to any one of claims 20 to 25, or the composition according to any one of claims 21 to 26 The kit.

27. A method for screening artificial circular RNAs comprising two or more hybridization regions capable of disrupting one or more target disruption structures of one or more RNA fragments by hybridization, wherein the target disruption structure is iii. A first region having at least a hairpin loop before or after the second region of an unpaired nucleotide; and iv. Defined as comprising at least one target hybridization region containing at least 2 nucleotides, preferably 3 nucleotides or more, single-stranded regions before or after a double-stranded region of at least 5 nucleotides, preferably 10 nucleotides or more. The method described above is d) A step of identifying the two or more hybridization regions of the artificial circular RNA as regions having a total length of 7 to 100 nucleotides, preferably 10 to 50 nucleotides, wherein when hybridizing with the at least one target hybridization region, the energy of the hybridization between the two or more hybridization regions and the at least one target hybridization region is negative compared to the energy of the target disruption structure, thereby disrupting the one or more target disruption structures, and the two or more hybridization regions contained in the artificial circular RNA are identified by an RNA reverse folding tool such as NUPACK, RNAifold, or MoiRNAiFold. e) A step of designing an artificial circular RNA comprising two or more hybridization regions capable of disrupting one or more target disruption structures identified in step a), wherein the artificial circular RNA is 150 to 800 nucleotides long, preferably 200 to 600 nucleotides long, and f) A method comprising optionally selecting the artificial circular RNA capable of disrupting one or more of the target disruption structures designed in step b) by hybridization, and optionally packaging it into a product.

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