RNA molecule for use in controlled translation

Abstract

The present invention provides an RNA molecule comprising: an open reading frame comprising a sequence which encodes a desired protein; an aptamer domain able to bind with a protein (target protein) different from the desired protein; and a short-chain single-stranded nucleic acid target domain comprising a target sequence with which a short-chain single-stranded nucleic acid can bind.

Description

RNA molecules for use in controlled translation

[0001] This invention relates to RNA molecules in which the translation of gene products is controlled, particularly RNA molecules in which gene translation is activated or suppressed in response to the intracellular environment. Furthermore, this invention relates to a method for activating gene translation in a specific cell.

[0002] mRNA has the characteristic of being able to directly induce protein expression in vivo without genome insertion. During the COVID-19 pandemic, vaccines utilizing mRNA technology have already been put into practical use, and it is expected to be a very useful modality in drug discovery.

[0003] When mRNA is administered to living organisms, carriers such as lipid nanoparticles (LNPs) are often used. LNPs are delivered non-selectively to cell types in the body, and mRNA efficiently induces protein expression in almost all cells. The selectivity of protein expression by mRNA is being studied.

[0004] Protein expression in living organisms is regulated by various mechanisms. For example, microRNAs (miRNAs, miRs), a type of functional non-coding RNA (ncRNA), are known to suppress the expression of various proteins by partially complementarily binding to multiple mRNA sequences and inhibiting mRNA translation. Research has been conducted on the use of miRNAs for cell-type selective expression of proteins via mRNA. For example, it has been reported that the regulation of protein expression of transgenes can be made dependent on cell-type specific miRNAs (Patent Documents 1-6 and Non-Patent Document 1).

[0005] Furthermore, there is one example of research that involves creating RNA containing an aptamer domain and regulating gene product expression through structural changes in the aptamer domain itself caused by ligand binding to the aptamer domain (Patent Document 7).

[0006] miRNAs exist in many organisms, and many types of miRNAs have been reported. Much research has been done on the function and role of miRNAs, and ubiquitous expression of miRNAs has also been reported (Non-Patent Documents 2 and 3).

[0007] International Patent Application Publication WO2018 / 165536A1 International Patent Application Publication WO2009 / 066758A1 International Patent Application Publication WO2018 / 003779A1 International Patent Application Publication WO2012 / 056440A1 International Patent Application Publication WO2022 / 266083A2 International Patent Application Publication WO2016 / 040395A1 US Patent Application Publication US2009 / 0098561A1

[0008] Fujita, Y et al., Science Advances, 5 Jan 2022, Vol 8, Issue 1, DOI: 10.1126 / sciadv.abj1793de Rie, D. et al., Nat. Biotechnol, 35, 872-878 (2017). https: / / doi.org / 10.1038 / nbt.3947Patil, Arun H et al. al., GigaScience vol. 11 (2022): giac083. doi:10.1093 / gigascience / giac083

[0009] Current technologies for delivering nucleic acids to cells in the body make it difficult to selectively deliver them to tissues and cells other than specific organs such as the liver and lungs. Therefore, there is a concern that gene products may be expressed in tissues other than the target tissue during gene delivery into the body. Consequently, there is a need for technologies that control the expression of delivered genes as proteins by initiating translation only when nucleic acids are taken up by the target tissue or cells, or by suppressing protein expression in unwanted tissues while increasing expression only in specific cells.

[0010] The inventors investigated the creation of a protein translation control mechanism utilizing ncRNA and discovered that gene product expression can be controlled using proteins present in cells by utilizing RNA molecules containing aptamer domains, thus completing the present invention. This specification encompasses the following disclosures of the invention.

[0011] [A1] An RNA molecule comprising an open reading frame (ORF) containing a sequence encoding a protein of interest, an aptamer domain capable of binding to a protein different from the protein of interest (target protein), and a short single-stranded nucleic acid target domain containing a target sequence to which a short single-stranded nucleic acid can bind, wherein the number of nucleotides in the short single-stranded nucleic acid is 200 or less.

[0012] [A2] The RNA molecule according to [A1], wherein the aptamer domain binds to the target protein, thereby inhibiting the binding of the target sequence to the short single-stranded nucleic acid. [A3] The RNA molecule according to [A1] or [A2], wherein the short single-stranded nucleic acid target domain is contained in the aptamer domain, the short single-stranded nucleic acid target domain is adjacent to the aptamer domain, one or more nucleotides in the aptamer domain are included as part of the target sequence of the short single-stranded nucleic acid target domain, or one or more nucleotides in the target sequence of the short single-stranded nucleic acid target domain are included as part of the aptamer domain.

[0013] [A4] The RNA molecule according to any one of [A1] to [A3], wherein the sequence located at the 5' end of the aptamer domain is capable of forming a stem with the sequence located at the 3' end of the aptamer domain. [A5] The RNA molecule according to [A4], wherein the sequence located at the 5' end and the sequence located at the 3' end of the aptamer domain, which are capable of forming a stem, each independently have 3 to 10 nucleotides.

[0014] [A6] An RNA molecule according to any one of [A1] to [A5] in which one or more nucleotides located at the 5' end of the aptamer domain constitute a part of the target sequence. [A7] An RNA molecule according to any one of [A1] to [A5] in which three to seven nucleotides located at the 5' end of the aptamer domain constitute a part of the target sequence.

[0015] [A8] An RNA molecule according to any one of [A1] to [A5] in which one or more nucleotides located at the 3' end of the aptamer domain constitute a part of the target sequence. [A9] An RNA molecule according to any one of [A1] to [A5] in which three to seven nucleotides located at the 3' end of the aptamer domain constitute a part of the target sequence.

[0016] [A10] An RNA molecule according to any one of [A1] to [A9] wherein a portion of the target sequence contains nucleotides that form a stem within the aptamer domain. [A11] An RNA molecule according to any one of [A1] to [A9] wherein a portion of the target sequence in the aptamer domain contains nucleotides that form a stem within the aptamer domain.

[0017] [A12] The RNA molecule according to any one of [A1] to [A11], wherein the nucleotides constituting a portion of the target sequence in the aptamer domain include nucleotides that do not form a stem within the aptamer domain.

[0018] [A13] An RNA molecule according to any one of [A1] to [A11] in which 3 to 7 nucleotides in the aptamer domain constitute a part of the target sequence. [A14] An RNA molecule according to any one of [A1] to [A5] in which the aptamer domain and the short single-stranded nucleic acid target domain are directly linked without overlap.

[0019] [A15] The RNA molecule according to [A14], wherein the 5' end of the aptamer domain and the 3' end of the target sequence of the short single-stranded nucleic acid target domain are directly linked without overlap. [A16] The RNA molecule according to [A14], wherein the 3' end of the aptamer domain and the 5' end of the target sequence of the short single-stranded nucleic acid target domain are directly linked without overlap.

[0020] [A17] An RNA molecule according to any one of [A1] to [A16], wherein the short single-stranded nucleic acid that can bind to the target sequence is an RNA having one of the sequence numbers: 125-134, 141, 142, or 144. [A18] An RNA molecule according to any one of [A1] to [A17], wherein the short single-stranded nucleic acid that can bind to the target sequence is an endogenous RNA selected from ncRNA, small RNA, and miRNA.

[0021] [A19] An RNA molecule according to any one of [A1] to [A16], wherein the short single-stranded nucleic acid capable of binding to the target sequence is exogenous RNA or exogenous DNA. [A20] An RNA molecule according to any one of [A1] to [A19], wherein the target protein capable of binding to the aptamer domain is a protein present in cells.

[0022] [A21] An RNA molecule according to any one of [A1] to [A20], wherein the target protein that can bind to the aptamer domain is a protein present in the cytoplasm. [A22] An RNA molecule according to any one of [A1] to [A20], wherein the target protein that can bind to the aptamer domain is the intracellular domain of a protein present in the cell membrane.

[0023] [A23] An RNA molecule according to any one of [A1] to [A22], wherein the portion other than the sequence encoding the target protein is an untranslated region (UTR). [A24] An RNA molecule according to any one of [A1] to [A23], wherein the aptamer domain and the short single-stranded nucleic acid target domain are located at the 3' end of the sequence encoding the target protein. [A25] An RNA molecule according to any one of [A1] to [A24], wherein the aptamer domain and the short single-stranded nucleic acid target domain are located at the 5' end of the sequence encoding the target protein. [A26] An RNA molecule according to any one of [A1] to [A25], which is a linear single-stranded RNA molecule.

[0024] [A27] An RNA molecule according to [A26], containing a poly(A) sequence at its 3' end. [A28] An RNA molecule according to [A26] or [A27], containing a CAP structure at its 5' end. [A29] An RNA molecule according to any of [A1] to [A25], which is a circular single-stranded RNA molecule. [A29-1] An RNA molecule according to [A29], further containing a CAP structure. [A29-2] An RNA molecule according to [A29] or [A29-1], further containing a poly(A) sequence. [A29-3] An RNA molecule according to any one of [A23] to [A29-2], further containing an IRES in its untranslated region.

[0025] [A30] An RNA molecule containing a modified nucleotide, as described in any of [A1] to [A29-3]. [A31] An RNA molecule as described in any of [A1] to [A30], wherein the translation of a target protein is inhibited when a short single-stranded nucleic acid binds to the target sequence of the short single-stranded nucleic acid target domain.

[0026] [A32] An RNA molecule according to any one of [A1] to [A31], wherein the target sequence is cleaved by the binding of a short single-stranded nucleic acid.

[0027] [A33] The RNA molecule according to any one of [A1] to [A32], wherein the aptamer domain has 20 to 100 nucleotides. [A34] The target sequence is a sequence that is completely complementary to any one of the sequences of SEQ ID NOs: 125 to 134, 141, 142, 144, or a sequence that is completely complementary to the portion starting from the second nucleotide and ending at the eighth nucleotide of any one of the sequences of SEQ ID NOs: 125 to 134, 141, 142, 144, and has a sequence that is incompletely complementary to the sequence starting from the ninth nucleotide. The RNA molecule according to any one of [A1] to [A33].

[0028] [A35] The RNA molecule according to any one of [A1] to [A34], wherein the short single-stranded nucleic acid capable of binding to the target sequence has any one of the sequences of SEQ ID NOs: 125 to 134. [A36] The RNA molecule according to any one of [A1] to [A35], wherein the sequence encoding the target protein has a sequence length of 20 kb or less.

[0029] [A37] The RNA molecule according to any one of [A1] to [A36], wherein the expression of the target protein is promoted by the binding of the aptamer domain to the target protein in the cell. [A38] A linear single-stranded RNA molecule, wherein the aptamer domain is included in the 3'UTR. The RNA molecule according to any one of [A1] to [A28] and [A30] to [A37].

[0030] [A39] The RNA molecule according to any one of [A1] to [A38], wherein the short single-stranded nucleic acid target domain contains only the target sequence. [A40] The RNA molecule according to [A19], wherein the short single-stranded nucleic acid is a single strand of shRNA or siRNA. [A41] The RNA molecule according to any one of [A1] to [A40], wherein the number of nucleotides constituting the short single-stranded nucleic acid is 20 to 40.

[0031] [A42] The short single-stranded nucleic acid is short single-stranded RNA or short single-stranded DNA, and the RNA molecule according to any one of [A1] to [A41]. [A43] A DNA molecule having a sequence from which a linear RNA molecule according to any one of [A1] to [A28] or any one of [A30] to [A41] can be obtained by transcription. [A44] A DNA molecule having a sequence from which a circular RNA molecule according to any one of [A1] to [A25] or any one of [A29] to [A41] can be obtained by transcription and cyclization reaction.

[0032] [B1] A composition comprising the RNA molecule according to any one of [A1] to [A42] or the DNA molecule according to any one of [A43] to [A44]. [B2] The composition according to [B1], further comprising a short single-stranded nucleic acid or a short single-stranded nucleic acid precursor, and the short single-stranded nucleic acid or the short single-stranded nucleic acid produced from the short single-stranded nucleic acid precursor can bind to the target sequence.

[0033] [B3] The composition according to [B2], wherein the short single-stranded nucleic acid precursor is shRNA or siRNA. [B4] The composition according to any one of [B1] to [B3], further comprising a carrier. [B5] A plasmid incorporated with the DNA molecule according to [A43]. [B6] A vector incorporated with the DNA molecule according to [A43].

[0034] [B7] A vector incorporated with the DNA molecule according to [A43] and a DNA molecule encoding a precursor of a short single-stranded nucleic acid, wherein the short single-stranded nucleic acid can bind to the target sequence of the short single-stranded nucleic acid target domain. [B8] The vector according to [B7], wherein the short single-stranded nucleic acid precursor is shRNA or siRNA.

[0035] A composition comprising a vector as described in any of [B9], [B6], to [B8]. A nucleic acid complex comprising an RNA molecule as described in any of [B9-1], [A1], to [A42] or a DNA molecule as described in any of [A43], to [A44], and lipid nanoparticles (LNPs) or liposomes. [B9-2] The nucleic acid complex according to [B9-1], wherein the lipid nanoparticles (LNPs) comprise a cationic lipid, a polyethylene glycol (PEG) modified lipid, a neutral lipid, and a sterol. [B10] A composition according to any of [B1], to [B4], and [B9], or a nucleic acid complex according to any of [B9-1], to [B9-2], for use in the treatment or prevention of a disease. [B11] The composition or nucleic acid complex according to [B10], wherein the disease is selected from cancer, infectious diseases, genetic diseases, immune diseases, mental diseases, cardiovascular diseases, respiratory diseases, digestive diseases, neurological diseases, endocrine diseases, metabolic diseases, skin diseases, inflammatory diseases, musculoskeletal diseases, and lifestyle-related diseases. [B12] The composition or nucleic acid complex according to [B10] or [B11], wherein the disease is a genetic disease.

[0036] [B13] The composition or nucleic acid complex according to any one of [B10] to [B12], wherein the disease is a disease caused by a protein deficiency, low expression, or low activity. [B14] The composition or nucleic acid complex according to [B10], wherein the disease is characterized by the presence of a target protein in a cell, and the target protein has a therapeutic effect against the disease in the cell. [B15] The composition according to any one of [B10] to [B14], further comprising a carrier containing the RNA molecule or the DNA molecule. [B16] The composition according to [B15], wherein the carrier is selected from lipid nanoparticles (LNPs), liposomes, polymer nanoparticles, dendrimers, exosomes, viral vectors, peptide carriers, and inorganic nanoparticles.

[0037] A method for expressing a target protein in a cell, comprising introducing an RNA molecule described in any of [C1] [A1] to [A42] or a DNA molecule described in [A43] into a cell in which the target protein of the aptamer domain is present. The method according to [C1], further comprising introducing an RNA molecule described in any of [C2] [A1] to [A42] or a DNA molecule described in [A43] into a cell in which the target protein of the aptamer domain is not present, wherein the expression level of the target protein in the cell is less than the expression level in a cell in which the target protein of the aptamer domain is present. A method for controlling the expression of a target protein, comprising introducing an RNA molecule described in any of [C3] [A1] to [A42] or a DNA molecule described in [A43] into a cell.

[0038] [D1] A kit for controlling protein translation comprising (a) and (b) below: (a) an RNA molecule as described in any of [A1] to [A42] or a DNA molecule as described in [A43]; (b) the short single-stranded nucleic acid, or a short single-stranded nucleic acid precursor capable of producing the short single-stranded nucleic acid by cleavage or dissociation within a cell. [D2] The kit according to [D1], wherein the short single-stranded nucleic acid precursor is shRNA or siRNA.

[0039] A method for detecting the presence of a target protein in a cell, comprising introducing an RNA molecule described in any of [E1], [A1] to [A42], or a DNA molecule described in [A43] into a cell, wherein: the target protein is a reporter protein, and the presence of the target protein is detected by measuring the expression level of the reporter protein compared to a control cell in which the target protein is absent. [E2] The method according to [E1], wherein the target protein is a protein specifically expressed in senescent cells, cancer cells, or virus-infected cells, and the state of the cell is determined using the expression of the reporter protein as an indicator.

[0040] This disclosure provides RNA molecules that promote the expression of a target protein only in cells where a specific target protein is present, and inhibit the expression of the target protein in the absence of the specific target protein, compositions comprising such RNA molecules, DNA molecules encoding such RNA molecules, and plasmids / vectors.

[0041] Figure 1 shows the FACS measurement results of cells introduced with Control EGFP in Example 2. The histograms of TagBFP fluorescence values ​​for both the sample containing mRNA-transfected cells and the untreated HeLa cell sample are shown either superimposed or individually. (a) is a superimposed figure of the histograms of TagBFP fluorescence values ​​for the sample containing mRNA-transfected cells and the untreated HeLa cell sample, with the dotted line representing untreated HeLa cells and the solid line representing mRNA-transfected cells. (b) is a figure of the histogram of TagBFP fluorescence values ​​for the sample containing mRNA-transfected cells, and (c) is a figure of the histogram of TagBFP fluorescence values ​​for the untreated HeLa cell sample. In figures (a), (b), and (c), the vertical axis is the cell count and the horizontal axis is the fluorescence intensity of TagBFP. The gating settings for cells separated from mRNA-transfected cells as BFP(-), BFP low, and BFP high are as shown in the figure, and the same gating settings were applied to cells into which each RNA construct was introduced. Figure 2 shows the structure of the mRNA constructs used in Example 2. The left side of each mRNA construct is the 5' end, and the right side is the 3' end. "AAA..." represents the poly-A sequence. Control EGFP contains the sequence encoding EGFP in the ORF, but does not contain the target sequence or aptamer domain. EGFP_Tg21 contains the sequence encoding EGFP in the ORF and the miR21 target sequence in the 3'UTR. EGFP_CDmini_Tg21 contains an EGFP-coding sequence in its ORF, and its 3'UTR contains a miR21 target sequence and an aptamer domain (L7Ae binding sequence, also called CDmini) that binds to L7Ae. The miR21 target sequence and the aptamer domain are adjacent to each other, and the aptamer domain and miR21 target sequence are positioned in the 5' to 3' direction. Figure 3 is a graph showing the normalized EGFP fluorescence intensity of each RNA construct in the BFP(-) cell fraction, the BFP low cell fraction, and the BFP high cell fraction.The horizontal axis displays the name of each RNA construct, and the vertical axis shows the normalized EGFP fluorescence intensity. White bars represent the cell fraction with BFP(-), grid-patterned bars represent the cell fraction with BFP low, and black bars represent the cell fraction with BFP high. Figure 4 is a graph showing the L7Ae-dependent EGFP ON / OFF ratio for each RNA construct. The horizontal axis displays the name of each RNA construct, and the vertical axis shows the L7Ae-dependent EGFP ON / OFF ratio. Figure 5 shows the FACS measurement results of cells into which Control EGFP was introduced in Example 3. Histograms of TagBFP fluorescence values ​​are displayed either overlaid or individually for both the sample containing mRNA-transfected cells and the untreated HeLa cell sample. (a) is a figure showing the overlay of histograms of TagBFP fluorescence values ​​for a sample containing mRNA-transfected cells and an untreated HeLa cell sample, with the dotted line representing untreated HeLa cells and the solid line representing mRNA-transfected cells. (b) is a figure showing the histogram of TagBFP fluorescence values ​​for a sample containing mRNA-transfected cells, and (c) is a figure showing the histogram of TagBFP fluorescence values ​​for an untreated HeLa cell sample. In figures (a), (b), and (c), the vertical axis is the cell count and the horizontal axis is the fluorescence intensity of TagBFP. The gating settings for cells separated from mRNA-transfected cells as BFP(-), BFP low, and BFP high are as shown in the figure, and the same gating setting is used for cells into which each RNA construct has been introduced. Figure 6 is a figure showing the structure of the mRNA construct used in Example 3. The left side of each mRNA construct is the 5' end and the right side is the 3' end. "AAA…" represents the poly-A sequence. Control EGFP contains the EGFP-coding sequence in its ORF but does not contain the target sequence or aptamer domain. EGFP_Tg21 contains the EGFP-coding sequence in its ORF and the miR21 target sequence in its 3'UTR.Each EGFP_ASn_CD_Tg21 (where n is an integer from 3 to 9) contains an EGFP-coding sequence in its ORF, a miR21 target sequence and an aptamer domain in its 3'UTR, the n nucleotides at the 3' end of the aptamer domain are also the n nucleotides at the 5' end of the miR21 target sequence, the n nucleotides at the 5' end of the aptamer domain have a sequence complementary to the n nucleotides at the 3' end of the aptamer domain and are capable of forming a stem, and the portion of the aptamer domain other than the stem-forming nucleotides is the CDminiΔstem. Each EGFP_Tg21_CD_ASm (where m is an integer from 3 to 8) contains an EGFP-coding sequence in its ORF, a miR21 target sequence and an aptamer domain in its 3'UTR, m nucleotides at the 5' end of the aptamer domain are also m nucleotides at the 3' end of the miR21 target sequence, and m nucleotides at the 3' end of the aptamer domain have a complementary sequence to the m nucleotides at the 5' end of the aptamer domain, allowing for stem formation. The portion of the aptamer domain other than the stem-forming nucleotides is the CDminiΔstem. Figure 7 is a graph showing the normalized EGFP fluorescence intensity of each RNA construct in the BFP(-) cell fraction, BFP low cell fraction, and BFP high cell fraction. The horizontal axis displays the name of the RNA construct, and the vertical axis shows the normalized EGFP fluorescence intensity. The white bars represent the BFP(-) cell fraction, the grid-patterned bars represent the BFP low cell fraction, and the black bars represent the BFP high cell fraction. Figure 8 is a graph showing the L7Ae-dependent EGFP ON / OFF ratio for each RNA construct. The horizontal axis displays the name of the RNA construct, and the vertical axis shows the L7Ae-dependent EGFP ON / OFF ratio. Figure 9 shows the FACS measurement results of cells into which G_TgSh was introduced in Example 4. Histograms of TagBFP fluorescence values ​​are displayed either overlaid or individually for both the sample containing mRNA-transfected cells and the untreated HeLa cell sample.(a) is a figure showing the overlay of histograms of TagBFP fluorescence values ​​for a sample containing mRNA-transfected cells and an untreated HeLa cell sample, with the dotted line representing untreated HeLa cells and the solid line representing mRNA-transfected cells. (b) is a figure showing the histogram of TagBFP fluorescence values ​​for a sample containing mRNA-transfected cells, and (c) is a figure showing the histogram of TagBFP fluorescence values ​​for an untreated HeLa cell sample. In figures (a), (b), and (c), the vertical axis represents the cell count and the horizontal axis represents the fluorescence intensity of TagBFP. The gating settings for cells separated from mRNA-transfected cells as BFP(-), BFP low, and BFP high are as shown in the figure, and the same gating setting was used for cells into which each RNA construct was introduced. Figure 10 is a figure showing the structure of the mRNA construct used in Example 4. The left side of each mRNA construct is the 5' end and the right side is the 3' end. "AAA…" represents the poly-A sequence. G_TgSh contains an EGFP-coding sequence in its ORF and an shRNA-binding sequence in its 3'UTR. G_ASn_CD_Tgsh (where n is an integer from 4 to 6) each contains an EGFP-coding sequence in its ORF and an shRNA-binding sequence and an aptamer domain in its 3'UTR, where the n nucleotides at the 3' end of the aptamer domain are also the n nucleotides at the 5' end of the shRNA-binding sequence, and the n nucleotides at the 5' end of the aptamer domain have a complementary sequence to the n nucleotides at the 3' end of the aptamer domain, allowing them to form a stem, and the portion of the aptamer domain other than the stem-forming nucleotides is the CDminiΔstem. G_Tgsh_CD_AS6 contains an EGFP-coding sequence in its ORF, an shRNA-binding sequence and an aptamer domain in its 3'UTR, the six nucleotides at the 5' end of the aptamer domain are also the six nucleotides at the 3' end of the shRNA-binding sequence, the six nucleotides at the 3' end of the aptamer domain have a complementary sequence to the six nucleotides at the 5' end of the aptamer domain and are capable of forming a stem, and the portion of the aptamer domain other than the stem-forming nucleotides is the CDminiΔstem.Figure 11 is a graph showing the normalized EGFP fluorescence intensity for each RNA construct in the BFP(-), BFP low, and BFP high cell fractions. The horizontal axis displays the name of the RNA construct, and the vertical axis shows the normalized EGFP fluorescence intensity. White bars represent the BFP(-) cell fraction, grid-patterned bars represent the BFP low cell fraction, and black bars represent the BFP high cell fraction. "no shRNA" indicates samples without co-introduction of shRNA, and "+shRNA" indicates samples with co-introduction of shRNA. Figure 12 is a graph showing the L7Ae-dependent EGFP ON / OFF ratio for each RNA construct. The horizontal axis displays the name of the RNA construct and whether or not shRNA was co-introduced, and the vertical axis shows the L7Ae-dependent EGFP ON / OFF ratio. "no shRNA" indicates samples without co-introduction of shRNA, and "+shRNA" indicates samples with co-introduction of shRNA. Figure 13 shows the FACS measurement results of cells into which G_TgLet7a_shuffle from Example 5 was introduced. Histograms of TagBFP fluorescence values ​​are shown overlaid or individually for both the sample containing mRNA-transfected cells and the untreated HeLa cell sample. (a) is a figure showing the overlaid histograms of TagBFP fluorescence values ​​for the sample containing mRNA-transfected cells and the untreated HeLa cell sample, with the dotted line representing untreated HeLa cells and the solid line representing mRNA-transfected cells. (b) is a figure showing the histogram of TagBFP fluorescence values ​​for the sample containing mRNA-transfected cells, and (c) is a figure showing the histogram of TagBFP fluorescence values ​​for the untreated HeLa cell sample. In figures (a), (b), and (c), the vertical axis is the cell count and the horizontal axis is the TagBFP fluorescence intensity. The gating settings for cells separated from mRNA-transfected cells as BFP(-), BFP low, and BFP high are as shown in the figure, and the same gating settings were applied to cells into which each RNA construct was introduced. Figure 14 shows the structure of the mRNA construct used in Example 5.The left side of each mRNA construct is the 5' end, and the right side is the 3' end. "AAA..." represents the poly-A sequence. G_TgLet7a contains the sequence encoding EGFP in its ORF and the let7a target sequence (let7a binding sequence) in its 3'UTR. G_TgLet7a_shuffle contains the sequence encoding EGFP in its ORF and the shuffled let7a binding sequence (a sequence that cannot bind to let7a) in its 3'UTR. G_CD_TgLet7a contains the sequence encoding EGFP in its ORF and the CDminiΔstem and let7a target sequence in its 3'UTR, where the CDminiΔstem and let7a target sequence are adjacent and arranged in 5' to 3' order. Each G_ASn_CD_TgLet7a (where n is an integer from 4 to 7) contains an EGFP-coding sequence in its ORF, and includes a let7a target sequence and an aptamer domain in its 3'UTR. The n nucleotides at the 3' end of the aptamer domain are also the n nucleotides at the 5' end of the let7a target sequence, and the n nucleotides at the 5' end of the aptamer domain have a complementary sequence to the n nucleotides at the 3' end of the aptamer domain, allowing them to form a stem. The portion of the aptamer domain other than the stem-forming nucleotides is the CDminiΔstem. G_TgLet7a_CD contains an EGFP-coding sequence in its ORF, and includes a CDminiΔstem and a let7a target sequence in its 3'UTR. The CDminiΔstem and the let7a target sequence are adjacent, and the let7a target sequence and CDminiΔstem are arranged in the order from 5' to 3'. Each G_TgLet7a_CD_ASm (where m is an integer from 3 to 7) contains an EGFP encoding sequence in its ORF, a let7a target sequence and an aptamer domain in its 3'UTR, the m nucleotides at the 5' end of the aptamer domain are also the m nucleotides at the 3' end of the let7a target sequence, the m nucleotides at the 3' end of the aptamer domain have a sequence complementary to the m nucleotides at the 5' end of the aptamer domain and are capable of forming a stem, and the portion of the aptamer domain other than the stem-forming nucleotides is the CDminiΔstem.Figure 15 is a graph showing the normalized EGFP fluorescence intensity for each RNA construct in the BFP(-), BFP low, and BFP high cell fractions. The horizontal axis displays the name of the RNA construct, and the vertical axis displays the normalized EGFP fluorescence intensity. White bars represent the BFP(-) cell fraction, grid-patterned bars represent the BFP low cell fraction, and black bars represent the BFP high cell fraction. Figure 16 is a graph showing the L7Ae-dependent EGFP ON / OFF ratio for each RNA construct. The horizontal axis displays the name of the RNA construct, and the vertical axis displays the L7Ae-dependent EGFP ON / OFF ratio. Figure 17 shows the FACS measurement results of cells into which G_TgLet7a_shuffle from Example 6 was introduced. Histograms of TagBFP fluorescence values ​​are shown overlaid or individually for both samples containing mRNA-transfected cells and untreated HeLa cells. (a) is a figure showing the overlay of histograms of TagBFP fluorescence values ​​for a sample containing mRNA-transfected cells and an untreated HeLa cell sample, with the dotted line representing untreated HeLa cells and the solid line representing mRNA-transfected cells. (b) is a figure showing the histogram of TagBFP fluorescence values ​​for a sample containing mRNA-transfected cells, and (c) is a figure showing the histogram of TagBFP fluorescence values ​​for an untreated HeLa cell sample. In figures (a), (b), and (c), the vertical axis is the cell count and the horizontal axis is the fluorescence intensity of TagBFP. The gating settings for cells separated from mRNA-transfected cells as BFP(-), BFP low, and BFP high are as shown in the figure, and the same gating setting is used for cells into which each RNA construct has been introduced. Figure 18 is a figure showing the structure of the mRNA construct used in Example 6. The left side of each mRNA construct is the 5' end and the right side is the 3' end. "AAA..." represents the poly-A sequence. G_TgLet7a contains the sequence encoding EGFP in its ORF and the let7a target sequence (let7a binding sequence) in its 3'UTR.G_TgLet7a_shuffle contains the sequence encoding EGFP in its ORF and a shuffled let7a binding sequence (a sequence that cannot bind to let7a) in its 3'UTR. G_MS2_TgLet7a contains the sequence encoding EGFP in its ORF and a MCP-binding aptamer domain (MS2) and a let7a target sequence in its 3'UTR. MS2 and the let7a target sequence are adjacent to each other by one base, and are positioned in the order from 5' to 3'. G_MS2Δstem_TgLet7a contains the sequence encoding EGFP in its ORF and a sequence with the stem of MS2 removed (MS2Δstem) and a let7a target sequence in its 3'UTR. MS2Δstem and the let7a target sequence are adjacent to each other, and are positioned in the order from 5' to 3'. Each G_ASn_MS2Δstem _TgLet7a (where n is an integer from 4 to 7) contains an EGFP-coding sequence in its ORF, a let7a target sequence and an aptamer domain in its 3'UTR, where the n nucleotides at the 3' end of the aptamer domain are also the n nucleotides at the 5' end of the let7a target sequence, and the n nucleotides at the 5' end of the aptamer domain have a complementary sequence to the n nucleotides at the 3' end of the aptamer domain, allowing them to form a stem, and the portion of the aptamer domain other than the stem-forming nucleotides is the MS2Δstem. G_TgLet7a_MS2 contains an EGFP-coding sequence in its ORF, and contains MS2 and a let7a target sequence in its 3'UTR, where MS2 and the let7a target sequence are adjacent with a 2-base link between them, and the let7a target sequence and MS2 are arranged in the order from 5' to 3'. Each G_TgLet7a_MS2Δstem_ASm (where m is an integer from 4 to 7) contains an EGFP encoding sequence in its ORF, a let7a target sequence and an aptamer domain in its 3'UTR, m nucleotides at the 5' end of the aptamer domain are also m nucleotides at the 3' end of the let7a target sequence, and m nucleotides at the 3' end of the aptamer domain have a sequence complementary to the m nucleotides at the 5' end of the aptamer domain, allowing them to form a stem, and the portion of the aptamer domain other than the stem-forming nucleotides is the MS2Δstem.Figure 19 is a graph showing the normalized EGFP fluorescence intensity for each RNA construct in the BFP(-), BFP low, and BFP high cell fractions. The horizontal axis displays the name of the RNA construct, and the vertical axis displays the normalized EGFP fluorescence intensity. White bars represent the BFP(-) cell fraction, grid-patterned bars represent the BFP low cell fraction, and black bars represent the BFP high cell fraction. Figure 20 is a graph showing the MCP-dependent EGFP ON / OFF ratio for each RNA construct. The horizontal axis displays the name of the RNA construct, and the vertical axis displays the MCP-dependent EGFP ON / OFF ratio. Figure 21 shows the FACS measurement results of cells into which G_TgLet7a_shuffle from Example 7 was introduced. Histograms of TagBFP fluorescence values ​​are shown overlaid or individually for both samples containing mRNA-transfected cells and untreated HeLa cell samples. (a) is a figure showing the overlay of histograms of TagBFP fluorescence values ​​for a sample containing mRNA-transfected cells and an untreated HeLa cell sample, with the dotted line representing untreated HeLa cells and the solid line representing mRNA-transfected cells. (b) is a figure showing the histogram of TagBFP fluorescence values ​​for a sample containing mRNA-transfected cells, and (c) is a figure showing the histogram of TagBFP fluorescence values ​​for an untreated HeLa cell sample. In figures (a), (b), and (c), the vertical axis is the cell count and the horizontal axis is the fluorescence intensity of TagBFP. The gating settings for cells separated from mRNA-transfected cells as BFP(-), BFP low, and BFP high are as shown in the figure, and the same gating setting was used for cells into which each RNA construct was introduced. Figure 22 shows the structure of the mRNA construct used in Example 7. The left side of each mRNA construct is the 5' end and the right side is the 3' end. "AAA..." represents a poly-A sequence. G_TgLet7a contains the sequence encoding EGFP in its ORF and the let7a target sequence (let7a binding sequence) in its 3'UTR.G_TgLet7a_shuffle contains an EGFP-coding sequence in its ORF and a shuffled let7a-binding sequence (a sequence that cannot bind to let7a) in its 3'UTR. G_OSnASm_CD_TgLet7a (where n is an integer from 1 to 16 and m is an integer from 4 to 8) contains an EGFP-coding sequence in its ORF and a let7a target sequence and an aptamer domain in its 3'UTR. The n+m nucleotides at the 3' end of the aptamer domain are also the n+m nucleotides at the 5' end of the let7a target sequence. The M nucleotides at the 5' end of the aptamer domain have a sequence complementary to the M nucleotides at the 3' end of the aptamer domain and are capable of forming a stem. The portion of the aptamer domain other than the stem-forming nucleotides consists of the CDminiΔstem and the N nucleotides at the 5' end of the let7a target sequence. Figure 23 is a graph showing the normalized EGFP fluorescence intensity for each RNA construct in the BFP(-), BFP low, and BFP high cell fractions. The horizontal axis displays the name of the RNA construct, and the vertical axis displays the normalized EGFP fluorescence intensity. White bars represent the BFP(-) cell fraction, grid-patterned bars represent the BFP low cell fraction, and black bars represent the BFP high cell fraction. Figure 24 is a graph showing the L7Ae-dependent EGFP ON / OFF ratio for each RNA construct. The horizontal axis displays the name of the RNA construct, and the vertical axis displays the L7Ae-dependent EGFP ON / OFF ratio. Figure 25 shows the FACS measurement results of cells into which G_TgLet7a_shuffle from Example 8 was introduced. Histograms of TagBFP fluorescence values ​​are shown overlaid or individually for both samples containing mRNA-transfected cells and untreated HeLa cell samples. (a) is a figure showing the overlay of histograms of TagBFP fluorescence values ​​for a sample containing mRNA-transfected cells and a sample of untreated HeLa cells, with the dotted line representing untreated HeLa cells and the solid line representing mRNA-transfected cells.(b) is a histogram of TagBFP fluorescence values ​​for a sample containing mRNA-transfected cells, and (c) is a histogram of TagBFP fluorescence values ​​for an untreated HeLa cell sample. In figures (a), (b), and (c), the vertical axis represents the cell count, and the horizontal axis represents the fluorescence intensity of TagBFP. The gating settings for cells separated from mRNA-transfected cells as BFP(-), BFP low, and BFP high are as shown in the figures, and the same gating settings were applied to cells into which each RNA construct was introduced. Figure 26 is a graph showing the normalized EGFP fluorescence intensity in the BFP(-), BFP low, and BFP high cell fractions for each RNA construct. The horizontal axis displays the name of the RNA construct, and the vertical axis shows the normalized EGFP fluorescence intensity. The white bars represent the cell fraction with BFP(-), the grid-patterned bars represent the cell fraction with BFP low, and the black bars represent the cell fraction with BFP high. Figure 27 is a graph showing the L7Ae-dependent EGFP ON / OFF ratio for each RNA construct. The horizontal axis displays the name of the RNA construct, and the vertical axis shows the L7Ae-dependent EGFP ON / OFF ratio. Figure 28 shows the FACS measurement results of cells into which G_TgLet7a_shuffle from Example 9 was introduced. Histograms of TagBFP fluorescence values ​​are shown overlaid or individually for each sample, including a sample containing mRNA-transfected cells and an untreated HeLa cell sample. (a) is a graph showing the overlaid histograms of TagBFP fluorescence values ​​for a sample containing mRNA-transfected cells and an untreated HeLa cell sample, with the dotted line representing untreated HeLa cells and the solid line representing mRNA-transfected cells. (b) is a histogram of TagBFP fluorescence values ​​for a sample containing mRNA-transfected cells, and (c) is a histogram of TagBFP fluorescence values ​​for an untreated HeLa cell sample. In figures (a), (b), and (c), the vertical axis represents the cell count, and the horizontal axis represents the fluorescence intensity of TagBFP.The gating settings for cells separated as BFP(-), BFP low, and BFP high from mRNA-transfected cells are as shown in the figure, and the same gating settings were applied to cells into which each RNA construct was introduced. Figure 29 is a graph showing the normalized EGFP fluorescence intensity in the BFP(-), BFP low, and BFP high cell fractions for each RNA construct in Example 9. The horizontal axis displays the name of the RNA construct, and the vertical axis shows the normalized EGFP fluorescence intensity. White bars represent the BFP(-) cell fraction, grid-patterned bars represent the BFP low cell fraction, and black bars represent the BFP high cell fraction. Figure 30 is a graph showing the L7Ae-dependent EGFP ON / OFF ratio for each RNA construct in Example 9. The horizontal axis displays the name of the RNA construct, and the vertical axis shows the L7Ae-dependent EGFP ON / OFF ratio. Figure 31 shows the FACS measurement results of cells into which G_TgLet7a_shuffle from Example 10 was introduced. Histograms of TagBFP fluorescence values ​​are shown overlaid or individually for both the sample containing mRNA-transfected cells and the untreated HeLa cell sample. (a) is a figure showing the overlaid histograms of TagBFP fluorescence values ​​for the sample containing mRNA-transfected cells and the untreated HeLa cell sample, with the dotted line representing untreated HeLa cells and the solid line representing mRNA-transfected cells. (b) is a figure showing the histogram of TagBFP fluorescence values ​​for the sample containing mRNA-transfected cells, and (c) is a figure showing the histogram of TagBFP fluorescence values ​​for the untreated HeLa cell sample. In figures (a), (b), and (c), the vertical axis is the cell count and the horizontal axis is the fluorescence intensity of TagBFP. The gating settings for cells separated from mRNA-transfected cells as BFP(-), BFP low, and BFP high are as shown in the figure, and the same gating settings were applied to cells into which each RNA construct was introduced.Figure 32 is a graph showing the normalized EGFP fluorescence intensity in the BFP(-), BFP low, and BFP high cell fractions for each RNA construct in Example 10. The horizontal axis displays the name of the RNA construct, and the vertical axis shows the normalized EGFP fluorescence intensity. White bars represent the BFP(-) cell fraction, grid-patterned bars represent the BFP low cell fraction, and black bars represent the BFP high cell fraction. Figure 33 is a graph showing the CAXII-TagBFP-L7Ae-dependent EGFP ON / OFF ratio for each RNA construct in Example 10. The horizontal axis displays the name of the RNA construct, and the vertical axis shows the CAXII-TagBFP-L7Ae-dependent EGFP ON / OFF ratio. Figure 34 shows the FACS measurement results of cells from Example 11 that were transfected with the plasmid encoding L7Ae-G4S-TagBFP and introduced with G_TgLet7a_shuffle. The histograms of TagBFP fluorescence values ​​for the sample containing mRNA-transfected cells and the untreated HeLa cell sample are shown either superimposed or individually. (a) is a superimposed figure of the histograms of TagBFP fluorescence values ​​for the sample containing mRNA-transfected cells and the untreated HeLa cell sample, with the dotted line representing untreated HeLa cells and the solid line representing mRNA-transfected cells. (b) is a figure of the histogram of TagBFP fluorescence values ​​for the sample containing mRNA-transfected cells, and (c) is a figure of the histogram of TagBFP fluorescence values ​​for the untreated HeLa cell sample. In figures (a), (b), and (c), the vertical axis is the cell count and the horizontal axis is the fluorescence intensity of TagBFP. The gating settings for cells separated from mRNA-transfected cells as BFP(-), BFP low, and BFP high are as shown in the figure, and the same gating settings were applied to cells into which each RNA construct was introduced.Figure 35 is a graph showing the normalized EGFP fluorescence intensity in the BFP(-) cell fraction, BFP low cell fraction, and BFP high cell fraction for a sample from Example 11 in which the plasmid encoding L7Ae-G4S-TagBFP was transfected and each RNA construct containing G_TgLet7a_shuffle, G_TgLet7a, CDmini, or CD sequence was introduced. The horizontal axis displays the name of the RNA construct, and the vertical axis displays the normalized EGFP fluorescence intensity. White bars represent the BFP(-) cell fraction, grid-patterned bars represent the BFP low cell fraction, and black bars represent the BFP high cell fraction. Figure 36 is a graph showing the L7Ae-dependent EGFP ON / OFF ratio for samples from Example 11 that were transfected with the plasmid encoding L7Ae-G4S-TagBFP and introduced with various RNA constructs containing G_TgLet7a_shuffle, G_TgLet7a, CDmini, or CD sequences. The horizontal axis displays the names of the RNA constructs, and the vertical axis shows the L7Ae-dependent EGFP ON / OFF ratio. Figure 37 is a graph showing the normalized EGFP fluorescence intensity in the BFP(-) cell fraction, BFP low cell fraction, and BFP high cell fraction for samples from Example 11 that were transfected with the plasmid encoding TagBFP-G4S-MCP and introduced with various RNA constructs containing G_TgLet7a_shuffle, G_TgLet7a, MS2, or MS2Δstem sequences. The horizontal axis displays the name of the RNA construct, and the vertical axis shows the normalized EGFP fluorescence intensity. White bars represent the cell fraction with BFP(-), grid-patterned bars represent the cell fraction with BFP low, and black bars represent the cell fraction with BFP high. Figure 38 is a graph showing the MCP-dependent EGFP ON / OFF ratio for samples from Example 11 that were transfected with a plasmid encoding TagBFP-G4S-MCP and introduced each RNA construct containing G_TgLet7a_shuffle, G_TgLet7a, MS2, or MS2Δstem sequences.The horizontal axis displays the name of the RNA construct, and the vertical axis shows the MCP-dependent ON / OFF ratio of EGFP. Figure 39 shows the FACS measurement results of cells co-transfected with G_TgLet7a_shuffle and miRNA mimic negative control from Example 12. Histograms of TagBFP fluorescence values ​​are shown overlaid or individually for each sample, including a sample containing cells co-transfected with mRNA and miRNA mimic and a sample of untreated HeLa cells. (a) is a figure showing the histograms of TagBFP fluorescence values ​​overlaid for a sample containing cells co-transfected with mRNA and miRNA mimic and a sample of untreated HeLa cells, with the dotted line representing untreated HeLa cells and the solid line representing cells co-transfected with mRNA and miRNA mimic. (b) is a histogram of TagBFP fluorescence values ​​for a sample containing cells co-transfected with mRNA and miRNA mimic, and (c) is a histogram of TagBFP fluorescence values ​​for an untreated HeLa cell sample. In figures (a), (b), and (c), the vertical axis represents the cell count and the horizontal axis represents the fluorescence intensity of TagBFP. The gating settings for cells separated as BFP(-), BFP low, and BFP high from cells co-transfected with mRNA and miRNA mimic are as shown in the figures, and the same gating settings were applied to cells into which each RNA construct was introduced. Figures 40-1 and 40-2 are graphs showing the normalized EGFP fluorescence intensity in the BFP(-), BFP low, and BFP high fractions of cells transfected with each RNA construct. The horizontal axis labels the names of the RNA constructs and the vertical axis represents the normalized EGFP fluorescence intensity. The white bars represent the cell fraction with BFP(-), the grid-patterned bars represent the cell fraction with BFP low, and the black bars represent the cell fraction with BFP high. Figure 40-1 shows the results of co-transfection with miRNA mimic negative control, and Figure 40-2 shows the results of co-transfection with miRNA mimic.Figures 41-1 and 41-2 are graphs showing the MCP-dependent EGFP ON / OFF ratio for each RNA construct. The horizontal axis displays the name of the RNA construct, and the vertical axis displays the MCP-dependent EGFP ON / OFF ratio. Figure 41-1 shows the results of co-administration with miRNA mimic negative control, and Figure 41-2 shows the results of co-administration with miRNA mimic. Figure 42 shows the FACS measurement results of cells co-transfected with G_TgLet7a_shuffle and miRNA mimic negative control in Example 13. Histograms of TagBFP fluorescence values ​​are shown overlaid or individually for each sample, including cells containing cells co-transfected with mRNA and miRNA mimic, and for an untreated HeLa cell sample. (a) is a figure overlaying histograms of TagBFP fluorescence values ​​for a sample containing cells co-transfected with mRNA and miRNA mimics and a sample of untreated HeLa cells, with the dotted line representing untreated HeLa cells and the solid line representing cells transfected with mRNA. (b) is a figure of the histogram of TagBFP fluorescence values ​​for a sample containing cells co-transfected with mRNA and miRNA mimics, and (c) is a figure of the histogram of TagBFP fluorescence values ​​for an untreated HeLa cell sample. In figures (a), (b), and (c), the vertical axis is the cell count and the horizontal axis is the fluorescence intensity of TagBFP. The gating settings for cells separated as BFP(-), BFP low, and BFP high from cells co-transfected with mRNA and miRNA mimics are as shown in the figure, and the same gating setting is used for cells into which each RNA construct has been introduced. Figures 43-1 and 43-2 are graphs showing the normalized EGFP fluorescence intensity in the BFP(-), BFP low, and BFP high fractions of cells transfected with each RNA construct. The horizontal axis displays the name of the RNA construct, and the vertical axis displays the normalized EGFP fluorescence intensity. White bars represent the BFP(-) cell fraction, grid-patterned bars represent the BFP low cell fraction, and black bars represent the BFP high cell fraction.Figure 43-1 shows the results of co-administration with a miRNA mimic negative control, and Figure 43-2 shows the results of co-administration with a miRNA mimic. Figures 44-1 and 44-2 are graphs showing the LS12-dependent ON / OFF ratio of EGFP for each RNA construct. The horizontal axis displays the name of the RNA construct, and the vertical axis shows the LS12-dependent ON / OFF ratio of EGFP. Figure 44-1 shows the results of co-administration with a miRNA mimic negative control, and Figure 44-2 shows the results of co-administration with a miRNA mimic. Figure 45 illustrates the design of the plasmid used for circular RNA preparation. As the circularization module, the 5' and 3' fragments of the intron and exon derived from Anabaena pre-tRNA, which are Group I introns, were used. In addition, to improve the circularization efficiency, complementary external homology arm (EHA) and internal homology arm (IHA) sequences were introduced before and after the intron and exon. A Coxackievirus B3 (CVB3)-derived IRES (Internal ribosome entry site) was used as the IRES element to initiate translation, and the sequence encoding eGFP was placed downstream of it. The 5'UTR and 3'UTR regions were appropriately inserted with miRNA Let7a target sequences (Let7a site) and MCP-binding sequences (MS2 motif). These RNA-encoding regions were placed downstream of the T7 promoter, where transcription is initiated by T7 RNA polymerase, and a NotI restriction enzyme recognition sequence was placed at the transcription aggregation site. Figure 46-1 is a part of Figure 46, which shows the designs and names of various circular RNAs used for translation elevation evaluation. Each RNA was circularized using PIE, and therefore the prepared circular RNAs contained ligated exons and IHAs at both ends. For each circular RNA, the UTR 5' to the IRES in the pre-circularized RNA transcribed from the plasmid is denoted here as the 5'UTR, and the UTR 3' to the eGFP is denoted as the 3'UTR.The 5'UTR and 3'UTR of each RNA may contain the Let7a microRNA complementary sequence (TgLet7a), the MS2 motif (MS2), the MS2 motif with the stem region removed (MS2ΔStem), and 4 or 5 nucleotides complementary to TgLet7a (AS4 or AS5). The names listed under Construct are the names of the circular RNAs. Figure 46-2 is a portion of Figure 46. Figure 46-3 is a portion of Figure 46. Figure 46-4 is the legend for Figure 46. Figure 47 shows the confirmation electrophoresis results of various circular RNAs purified by RNaseR treatment after preparation. The names of the various samples are as listed in Figure 46. In all samples, an upper band expected to be the circular RNA and a lower band expected to be its Nicked product were observed. Figure 48 is a graph showing the normalized EGFP fluorescence intensity of each RNA construct in HeLa cells, HeLa-MCP-low cells, and HeLa-MCP-high cells. The horizontal axis displays the name of each RNA construct, and the vertical axis shows the normalized EGFP fluorescence intensity. White bars represent the HeLa cell fraction, grid-patterned bars represent the HeLa-MCP-low cell fraction, and black bars represent the HeLa-MCP-high cell fraction. Figure 49 is a graph showing the ON / OFF ratio of MCP-dependent EGFP expression for each RNA construct. The horizontal axis displays the name of each RNA construct, and the vertical axis shows the rate of increase in MCP-dependent EGFP translation (ON / OFF ratio), calculated by dividing the normalized EGFP fluorescence intensity in HeLa-MCP-high cells by the normalized EGFP fluorescence intensity in HeLa cells. Figure 50 is a diagram showing the structure of the mRNA construct used in Example 16. The left side of each mRNA construct is the 5' end, and the right side is the 3' end. "AAA…" represents the poly(A) sequence. Furthermore, "5'UTR" represents the untranslated region on the 5' side, and "3'UTR" represents the untranslated region on the 3' side. G_TgLet7a contains the sequence encoding EGFP in its ORF, and the 3'UTR contains the let7a target sequence (let7a binding sequence).G_TgLet7a_shuffle contains an EGFP-coding sequence in its ORF and a shuffled let7a-binding sequence (a sequence that cannot bind to let7a) at the 3'UTR. sn_TgLet7a_MS2Δstem_AS3 or 4_G (where n is an integer from 9 to 49) each contains an EGFP-coding sequence in its ORF and includes a let7a target sequence and an aptamer domain at a base number n from the 5' end, where the 3 or 4 nucleotides at the 5' end of the aptamer domain are also the 3 or 4 nucleotides at the 3' end of the let7a target sequence, and the 3 or 4 nucleotides at the 3' end of the aptamer domain have a sequence complementary to the 3 or 4 nucleotides at the 5' end of the aptamer domain and are capable of forming a stem, and the portion of the aptamer domain other than the stem-forming nucleotides is the MS2Δstem. Figure 51 is a graph showing the normalized EGFP fluorescence intensity of each RNA construct in HeLa cells, HeLa-MCP-low cells, and HeLa-MCP-high cells. The horizontal axis displays the name of each RNA construct, and the vertical axis shows the normalized EGFP fluorescence intensity. White bars represent the HeLa cell fraction, grid-patterned bars represent the HeLa-MCP-low cell fraction, and black bars represent the HeLa-MCP-high cell fraction. Figure 52 is a graph showing the ON / OFF ratio of MCP-dependent EGFP expression for each RNA construct. The horizontal axis displays the name of each RNA construct, and the vertical axis shows the rate of increase in MCP-dependent EGFP translation (ON / OFF ratio), calculated by dividing the normalized EGFP fluorescence intensity in HeLa-MCP-high cells by the normalized EGFP fluorescence intensity in HeLa cells. Figure 53 is a graph showing the normalized EGFP fluorescence intensity for each construct-encoding plasmid DNA, both with and without co-introduction of the plasmid DNA encoding TagBFP-G4S-MCP-2 (SEQ ID NO: 261). The horizontal axis displays the name of each construct, and the vertical axis shows the normalized EGFP fluorescence intensity.The white bars represent the normalized EGFP fluorescence intensity when the plasmid DNA encoding TagBFP-G4S-MCP-2 was not co-introduced, and the black bars represent the normalized EGFP fluorescence intensity when TagBFP-G4S-MCP-2 was co-introduced. Figure 54 is a graph showing the ON / OFF ratio of MCP-dependent EGFP expression for the plasmid DNA encoding each construct. The horizontal axis displays the name of each construct, and the vertical axis shows the rate of increase in MCP-dependent EGFP translation (ON / OFF ratio), calculated by dividing the normalized EGFP fluorescence intensity under conditions with the addition of the plasmid DNA encoding TagBFP-G4S-MCP-2 by the normalized EGFP fluorescence intensity under conditions without the addition of the plasmid DNA encoding TagBFP-G4S-MCP-2.

[0042] Nucleic Acids In this specification, the term "nucleic acid" means a polymer (also called a polynucleotide) of nucleotides (nucleotide monomers). Nucleic acids may include, for example, deoxyribonucleic acid (DNA), ribonucleic acid (RNA), threose nucleic acid (TNA), glycol nucleic acid (GNA), peptide nucleic acid (PNA), locked nucleic acid (LNA; including LNA having a β-D-ribo structure, α-LNA having an α-L-ribo structure (a diastereomer of LNA), 2'-amino-LNA having 2'-amino functionalization, and 2'-amino-α-LNA having 2'-amino functionalization), ethylene nucleic acid (ENA), cyclohexenyl nucleic acid (CeNA), and / or chimeras and / or combinations thereof. Nucleic acids may be single-stranded or double-stranded.

[0043] RNA In this specification, the terms "RNA molecule" or "RNA" refer to nucleic acid molecules containing ribose sugar and comprising at least one base selected from the group consisting of adenine (A), guanine (G), cytosine (C), and uracil (U). RNA includes, but is not limited to, naturally occurring RNA, synthetic RNA, modified RNA, and their derivatives.

[0044] As used herein, “RNA” refers to ribonucleic acid, whether naturally occurring or unnatural. For example, RNA may contain one or more modified and / or unnatural components, such as nucleic acid bases, nucleosides, nucleotides, or linkers.

[0045] RNA molecules may have a single-stranded structure, and if an RNA molecule is single-stranded RNA, it may include a cap structure, a terminal nucleoside, a stem-loop, a poly(A) sequence, and / or a polyadenylation signal. RNA may have a nucleotide sequence that codes for a polypeptide of interest. For example, RNA may be messenger RNA (mRNA). Translation of mRNA that codes for a specific polypeptide, for example, in vivo translation of mRNA in mammalian cells, may produce the coded polypeptide. RNA may be selected from an unrestricted group consisting of small interfering RNA (siRNA), asymmetric interfering RNA (aiRNA), microRNA (miRNA), Dicer substrate RNA (dsRNA), small hairpin RNA (shRNA), mRNA, non-coding RNA (ncRNA), long non-coding RNA (lncRNA), and mixtures thereof. RNA molecules may have a double-stranded structure.

[0046] Circular RNA: RNA molecules may be circular RNA molecules. In this specification, "circular RNA" refers to ribonucleic acid (RNA) molecules having a bounded circular structure. Due to its circular structure, circular RNA has higher stability than typical linear RNA and is resistant to degradation by exonucleases. This term includes not only naturally occurring circular RNA but also artificially designed and synthesized circular RNA molecules.

[0047] Circular RNA may be a single-stranded RNA molecule. Circular RNA may also have an IRES (Internal Ribosome Entry Site, see Kieft et al., (2001) RNA 7(2):194-206). An IRES is an RNA secondary structure that enables translation initiation independent of the mRNA's 5' end. Specifically, IRES directly recruits ribosomes and promotes cap-independent translation initiation. Examples of IRESs include, specifically, Taura syndrome virus, Triatoma virus, Tyler's encephalomyelitis virus, Simian virus 40, Solenopsis invicta virus 1, Aphid virus, Reticuloendotheliosis virus, Human poliovirus 1, Brown marmorated stink bug enteric virus, Cassiopeia wasp virus, Human rhinovirus 2, Homalodisca coagulata virus-1, Human immunodeficiency virus type 1, Homalodisca virus-1, Himetobi P virus, Hepatitis C virus, Hepatitis A virus, Hepatitis GB virus, Foot-and-mouth disease virus, Human enterovirus 71, Equine rhinitis virus, and Ectropis Obliqua picorna-like virus, encephalomyocarditis virus (EMCV), Drosophila C virus, Crucifer tobamo virus, Cricket paralysis virus, Bovine viral diarrhea virus 1, Black queen bee disease virus, Aphid lethal paralysis virus, Avian encephalomyelitis virus, Acute honeybee paralysis virus, Hibiscus chlorotic ringspot virusvirus), classical swine fever virus, human FGF2, human SFTPAl, human AMLI / RUNXL, Drosophila antennapedia, human AQP4, human AT1R, human BAG-1, human BCL2, human BiP, human c-IAPl, human c-myc, human eIF4G, mouse NDST4L, human LEF1, mouse HIF1α, human n.myc, mouse Gtx, human p27kipl, human PDGF2 / c-sis, human p53, human Pim-1, mouse Rbm3, Drosophila reaper, canine scamper, Drosophila Ubx, Salivirus, Cosavirus, Parechovirus, human UNR, mouse UtrA, human VEGF-A, human XIAP, Drosophila hairless, yeast (S. Examples include cerevisiae)TFIID, yeast YAP1, human c-src, human FGF-1, salpicornavirus, turnip crinkle virus, aptamers for eIF4G, and IRES sequences of coxsackievirus B3 (CVB3) or coxsackievirus A (CVB1 / 2). Furthermore, examples of IRES include Taura syndrome virus, Triatoma virus, Theiler's encephalomyelitis virus, Simian virus 40, Solenopsis invicta virus 1, Rhopalosiphum padi virus, reticuloendotheliosis virus, human poliovirus 1, Plautia stali enteric virus, cashmere honeybee virus, human rhinovirus 2, Homalodisca coagulata virus-1, human immunodeficiency virus type 1, Homalodisca coagulata virus-1, Himetobi P virus, hepatitis C virus, hepatitis A virus, hepatitis GB virus, foot-and-mouth disease virus, human enterovirus 71, horse rhinovirus, and EctropisObliqua picorna-like virus, encephalomyocarditis virus (EMCV), Drosophila C virus, Brassicaceae tobamovirus, cricket paralysis virus, bovine viral diarrhea virus 1, black queen cell virus, aphid lethal paralysis virus, avian encephalomyelitis virus, acute honeybee paralysis virus, hibiscus chlorophyll ring spot virus, classical swine fever virus, human FGF2, human SFTPA1, human AML1 / RUNX1, Drosophila antennapedia, human AQP4, human AT1R, human BAG-1, human BCL2, human BiP, human c-IAPl, human c-myc, human eIF4G, mouse NDST4L, human LEF1, mouse HIF1 alpha, human n.myc, mouse Gtx, human p27kipl, human PDGF2 / c-sis, human p53, human Pim-l, mouse Rbm3, fruit fly reaper, dog Scamper, fruit fly Ubx, Salivirus, Cosavirus, Parechovirus, human UNR, mouse UtrA, human VEGF-A, human XIAP, fruit fly hairless, S. cerevisiae TFIID, S. cerevisiae YAP1, human c-src, human FGF-l, simian picornavirus, turnip cigar virus, aptamer for eIF4G, coxsackievirus B3 (CVB3) or coxsackievirus AIRES sequences derived from (CVB1 / 2) are cited. Furthermore, examples of IRES include Aalivirus, Ailurivirus, Ampivirus, Anativirus, Aphthovirus, Aquamavirus, Avihepatovirus, Avisivirus, Boosepivirus, Bopivirus, Caecilivirus, Cardiovirus, Cosavirus, Crahelivirus, Crohivirus, Danipivirus, Dicipivirus, Diresapivirus, Enterovirus, Erbovirus, Felipivirus, Fipivirus, Gallivirus, Gruhelivirus, Grusopivirus, Harkavirus, Hemipivirus, Hepatovirus, Hunnivirus, Kobuvirus, Kunsagivirus, Limnipivirus, Livupivirus, Ludopivirus, Malagasivirus, Marsupivirus, Megrivirus, and Mischivir. s, Mosavirus, Mupivirus, Myrropivirus, Orivirus, Oscivirus, Parabovirus, Parechovirus, Pasivirus, Passerivirus, Pemapivirus, Poecivirus, Potamipivirus, Pygoscepivirus, Rabovirus, Rafivirus, Rajidapivirus, Rohelivirus, Rosavirus, Sakobuvirus, Salivirus, Sapelovirus, Senecavirus, Shanbavirus, Sicinivirus, Symapivirus, Teschovirus, Torchivirus, Tottorivirus, Tremovirus, Tropivirus, Hepacivirus, Pegivirus, Pestivirus, Flavivirus, Taura syndrome virus, Triatoma virus, Theiler's encephalomyelitis virus, Simian virus 40, Solenopsis invicta virus 1, RhopalosiphumPADI virus, reticuloendotheliosis virus, human poliovirus 1, Plautia stali enteric virus, cashmere honeybee virus, human rhinovirus 2, Homalodisca coagulata virus-1, human immunodeficiency virus type 1, Himetobi P virus, hepatitis C virus, hepatitis A virus, hepatitis GB virus, foot-and-mouth disease virus, human enterovirus 71, equine rhinovirus, Ectropis Obliqua picorna-like virus, encephalomyocarditis virus, Drosophila C virus, human coxsackievirus B3, Brassicaceae tobamovirus, cricket paralysis virus, bovine viral diarrhea virus 1, black queen cell virus, aphid lethal paralysis virus, avian encephalomyelitis virus, acute honeybee paralysis virus, hibiscus chlorophyll ring spot virus, classical swine fever virus, human FGF2, human SFTPA1, human AML1 / RUNX1, Drosophila antennapedia, human AQP4, human AT1R, human BAG-1, human BCL2, human BiP, human c-IAP1, human c-myc, human eIF4G, mouse NDST4L, human LEF1, mouse HIF1 alpha, human n.myc, mouse Gtx, human p27kip1, human PDGF2 / c-sis, human p53, human Pim-1, mouse Rbm3, fruit fly reaper, dog Scamper, fruit fly Ubx, human UNR, mouse UtrA, human VEGF-A, human XIAP, fruit fly hairless, S. cerevisiae TFIID, S. cerevisiae YAP1, tobacco ecchi virus, turnip cigar virus, EMCV-A, EMCV-B, EMCV-Bf, EMCV-Cf, EMCV pEC9, Picovirnavirus, HCV QC64, Human Cosavirus E / D, Human Cosavirus F, Human Cosavirus JMY, Rhinovirus NAT001, HRV14, HRV89, HRVC-02, HRV-A21, Salivirus A SH1, Salivirus FHB, Salivirus NG-J1, Human Parechovirus 1, Crohivirus B, Yc-3, Rosavirus M-7, ShanbavirusA, Pasivirus A, Pasivirus A 2, Echovirus E14, Human Parechovirus 5, Aichi Virus, Hepatitis A Virus HA16, Phopivirus, CVA10, Enterovirus C, Enterovirus D, Enterovirus J, Human Pegivirus 2, GBV-C GT110, GBV-C K1737, GBV-C Iowa, Pegivirus A 1220, Pasivirus A 3, Sapelovirus, Rosavirus B, Bakunsa Virus, Tremovirus A, Swine Pasivirus 1, PLV-CHN, Pasivirus A, Sicinivirus, Hepacivirus K. Hepacivirus A. BVDV1. Border Disease Virus BVDV2. CSFV-PK15C. SF573 Dicistrovirus GUT, Salivavirus A CH, Salivavirus A SZ1, Salivavirus FHB, CVB3, CVB1, Echovirus 7 CVB5, EVA71, CVA3, CVA12, EV24 The current IRES switch is based on eIF4G.

[0048] IRES may include eukaryotic or cellular IRES in whole or in part. IRES may be derived from human genes, and such human genes may be any of the following: ABCF1, ABCG1, ACAD10, ACOT7, ACSS3, ACTG2, ADCYAP1, ADK, AGTR1, AHCYL2, AHI1, AKAP8L, AKR1A1, ALDH3A1, ALDOA, ALG13, AMMECR1L, ANGPTL4, ANK3, AOC3, AP4B1, AP4E1, APAF1, APBB1, APC, APH1A, APOBEC3D, APOM, APP, AQP4, ARHGAP36, ARL13B, ARMC8, ARMCX6, ARPC1A, ARPC2, ARRDC3, ASAP1, ASB3, ASB5, ASCL1, ASMTL, ATF2, ATF3, ATG4A, ATP5B, ATP6V0A1, ATXN3, AURKA, AURKA, AURKA, AURK A, B3GALNT1, B3GNTL1, B4GALT3, BAAT, BAG1, BAIAP2, BAIAP2L2, BAZ2A, BBX, BCAR1, BCL2, BCS1L, BET1, BID, BIRC2, BPGM, BPIFA2, BRINP2, BSG, BT N3A2, C12orf43, C14orf93, C17orf62, C1orf226, C21orf62, C2orf15, C4BPB, C4orf22, C9orf84, CACNA1A, CALCOCO2, CAPN11, CASP12, CASP8AP2, CAV1, CBX5, CCDC120, CCDC17, CCDC186, CCDC51, CCN1, CCND1, CCNT1, CD2BP2, CD9, CDC25C, CDC42, CDC7, CDCA7L, CDIP1, CDK1, CDK11A, CDKN1B, CE ACAM7, CEP295NL, CFLAR, CHCHD7, CHIA, CHIC1, CHMP2A, CHRNA2, CLCN3, CLEC12A, CLEC7A, CLECL1, CLRN1, CMSS1, CNIH1, CNR1, CNTN5, COG4, COMMD 1, COMMD5, CPEB1, CPS1, CRACR2B, CRBN, CREM, CRYBG1, CSDE1, CSF2RA, CSNK2A1, CSTF3, CTCFL, CTH, CTNNA3, CTNNB1, CTNNB1, CTNND1, CTSL, CUTA,CXCR5, CYB5R3, CYP24A1, CYP3A5, DAG1, DAP3, DAP5, DAXX, DCAF4, DCAF7, DCLRE1A, DCP1A 、DCTN1、DCTN2、DDX19B、DDX46、DEFB123、DGKA、DGKD、DHRS4、DHX15、DIO3、DLG1、DLL4、DMD UTR、DMD ex5, DMKN, DNAH6, DNAL4, DUSP13, DUSP19, DYNC1I2, DYNLRB2, D YRK1A, ECI2, ECT2, EIF1AD, EIF2B4, EIF4G1, EIF4G2, EIF4G3, EL ANE, ELOVL6, ELP5, EMCN, ENO1, EPB41, ERMN, ERVV-1, ESRRG, ET FB, ETFBKMT, ETV1, ETV4, EXD1, EXT1, EZH2, FAM111B, FAM157A, F AM213A, FBXO25, FBXO9, FBXW7, FCMR, FGF1, FGF1, FGF1A, FGF2 FGF2, FGF-9, FHL5, FMR1, FN1, FOXP1, FTH1, FUBP1, G3BP1, and GABBR 1, GALC, GART, GAS7, gastrin, GATA1, GATA4, GFM2, GHR, GJB2, GLI1, GLRA2, GMNN, GPAT3, GPATCH3, GPR137, GPR34, GPR55, GPR89A GPRASP1, GRAP2, GSDMB, GSTO2, GTF2B, GTF2H4, GUCY1B2, HAX1, HCST, HIGD1A, HIGD1B, HIPK1, HIST1H1C, HIST1H3H, HK1, HLA-D RB4, HMBS, HMGA1, HNRNPC, HOPX, HOXA2, HOXA3, HPCAL1, HR, HSP9 0AB1, HSPA1A, HSPA4L, HSPA5, HYPK, IFFO1, IFT74, IFT81, IGF1. IGF1R, IGF1R, IGF2, IL11, IL17RE, IL1RL1, IL1RN, IL32, IL6, ILF2, ILVBL, INSR, INTS13, IP6K1, ITGA4, ITGAE, KCNE4, KERA, KI AA0355, KIAA0895L, KIAA1324, KIAA1522, KIAA1683, KIF2C, KIZ KLHL31, KLK7, KRR1, KRT14, KRT17, KRT33A, KRT6A, KRTAP10-2KRTAP13-3、KRTAP13-4、KRTAP5-11、KRTCAP2、LACRT、LAMB1、LAMB3、LANCL1 、LBX2、LCAT、LDHA、LDHAL6A、LEF1、LINC-PINT、LMO3、LRRC4C、LRRC7、LRTOM T、LSM5、LTB4R、LYRM1、LYRM2、MAGEA11、MAGEA8、MAGEB1、MAGEB16、MAGEB3、 MAPT、MARS、MC1R、MCCC1、METTL12、METTL7A、MGC16025、MGC16025、MIA2、MIA 2、MITF、MKLN1、MNT、MORF4L2、MPD6、MRFAP1、MRPL21、MRPS12、MSI2、MSLN、M SN、MT2A、MTFR1L、MTMR2、MTRR、MTUS1、MYB、MYC、MYCL、MYCN、MYL10、MYL3、M YLK、MYO1A、MYT2、MZB1、NAP1L1、NAV1、NBAS、NCF2、NDRG1、NDST2、NDUFA7、N DUFB11、NDUFC1、NDUFS1、NEDD4L、NFAT5、NFE2L2、NFE2L2、NFIA、NHEJ1、NHP2 、NIT1、NKRF、NME1-NME2、NPAT、NR3C1、NRBF2、NRF1、NTRK2、NUDCD1、NXF2、N XT2、ODC1、ODF2、OPTN、OR10R2、OR11L1、OR2M2、OR2M3、OR2M5、OR2T10、OR4C 15、OR4F17、OR4F5、OR5H1、OR5K1、OR6C3、OR6C75、OR6N1、OR7G2、p53、P2RY4 、PAN2、PAQR6、PARP4、PARP9、PC、PCBP4、PCDHGC3、PCLAF、PDGFB、PDZRN4、PEL O、PEMT、PEMT、PFKM、PGBD4、PGLYRP3、PHLDA2、PHTF1、PI4KB、PIGC、PIM1、PK D2L1、PKM、PLCB4、PLD3、PLAY1、PLEKHB1、PLS3、PML、PNMA5、PNN、POC1A、PO C1B、POLD2、POLD4、POU5F1、PPIG、PQBP1、PRAME、PRPF4、PRR11、PRRT1、PRSS 8、PSMA2、PSMA3、PSMA4、PSMD11、PSMD4、PSMD6、PSME3、PSMG3、PTBP3、PTCH1、PTHLH, PTPRD, PUS7L, PVRIG, QPRT, RAB27A, RAB7B, RABGGTB, RAET1E, RALGDS, RALYL, RARB, RCVRN, REG3G, RFC5, RGL4, RGS19, RGS3, RHD, RINL, RIPOR2, RITA1, RMDN2, RNASE1, RNASE4, RNF4, RPA2, RPL17, RPL21, RPL26L1, RPL28, RPL29, RPL41, RPL9, RPS11, RPS13, RPS14, RRBP1, RSU1, RTP2, RUNX1, RUNX1T 1, RUNX1T1, RUNX2, RUSC1, RXRG, S100A13, S100A4, SAT1, SCHIP1, SCMH1, SEC14L1, SEMA4A, SERPINA1, SERPINB4, SERTAD3, SFTPD, SH3D19, SHC1, SHMT1, SHPRH, SIM1, SIRT5, SLC11A2, SLC12A4, SLC16A1, SLC25A3, SLC26A9, SLC5A11, SLC6A12, SLC6A19, SLC7A1, SLFN11, SLIRP, SMAD5, SMARCAD1, SMN1, SNCA , SNRNP200, SNRPB2, SNX12, SOD1, SOX13, SOX5, SP8, SPARCL1, SPATA12, SPATA31C2, SPN, SPOP, SQSTM1, SRBD1, SRC, SREBF1, SRPK2, SSB, SSB, SSBP1, ST3GAL6, STAB1, STAMBP, STAU1, STAU1, STAU1, STAU1, STK16, STK24, STK38, STMN1, STX7, SULT2B1, SYK, SYNPR, TAF1C, TAGLN, TANK, TAS2R40, TBC1D 15、TBXAS1、TCF4、TDGF1、TDP2、TDRD3、TDRD5、TESK2、THAP6、THBD、THTPA、TIAM2、TKFC、TKTL1、TLR10、TM9SF2、TMC6、TMCO2、TMED10、TMEM116、TMEM126A 、TMEM159、TMEM208、TMEM230、TMEM67、TMPRSS13、TMUB2、TNFSF4、TNIP3、TP53、TP53、TP73、TRAF1、TRAK1、TRIM31、TRIM6、TRMT1、TRMT2B、TRPM7、TRPM8、TSPEAR, TTC39B, TTLL11, TUBB6, TXLNB, TXNIP, TXNL1, TXNRD1, TYROBP, U2AF1, UBA1, UBE2D3, UBE2I, UBE2L3, U BE2V1, UBE2V2, UMPS, UNG, UPP2, USMG5, USP18, UTP14A, UTRN, UTS2, VDR, VEGFA, VEGFA, VEPH1, VIPAS39, VPS29, VSIG10L, WDHD1, WDR12, WDR4, WDR45, WDYHV1, WRAP53, XIAP, XPNPEP3, YAP1, YWHAZ, YY1AP1, ZBTB32, ZNF146, ZNF250, ZNF385A, ZNF408, ZNF410, ZNF423, ZNF43, ZNF502, ZNF512, ZNF513, ZNF580, ZNF609, ZNF707, or ZNRD1. Wild-type IRES sequences may also be modified and potentially effective in this invention. IRES sequences may be 100–1200 nucleotides long, 300–700 nucleotides long, or approximately 500–600 nucleotides long.

[0049] Circular RNA may form a ring by linking the nucleotides at the 5' and 3' ends of linear RNA. Circular RNA may also form a ring by chemical conjugation of components other than nucleotides (e.g., cap compounds).

[0050] Circular RNA can be produced by one of the following methods: splicing after transcription to create a circular RNA (a reaction that also occurs in the human body), ligation with RNA / DNA ligases, conjugation by chemical reactions, circularization with splicing ribozymes (group I introns, group II introns), or ligation within cells with intracellular ligases (such as RtcB). For methods of producing circular RNA, please refer to Obi et al. (Methods, Volume 196, December 2021, Pages 85-103), Sharma et al. (Funct Integr Genomics. 2024 Jun 26;24(4):117.), Lee et al. (Int J Mol Sci. 2024 Aug 30;25(17):9437.), Roth et al. (iScience. Volume 24, Issue 12, 17 December 2021, 103431), Mamot et al. (https: / / doi.org / 10.1101 / 2024.10.10.617555), and Chen et al. (Nature biotechnology. https: / / doi.org / 10.1038 / s41587-024-02393-y).

[0051] Circular RNA (circRNA) may further comprise a 5' cap structure (e.g., m7G cap, m7GpppN, etc.) or a functional equivalent thereof. While circular RNA typically features a closed ring structure lacking free 5' and 3' ends, it is possible to confer or associate a translation-initiating cap structure to such circular RNA using one of the following methods.

[0052] As a first method, circular RNA may have branched strands containing cap structures (branch structures) (see: Chen, H., Liu, D., Aditham, A. et al. Chemical and topological design of multicapped mRNA and capped circular RNA to augment translation. Nat Biotechnol 43, 1128-1143 (2025), Fukuchi, K., Nakashima, Y., Abe, N. et al. Internal cap-initiated translation for efficient protein production from circular mRNA. Nat Biotechnol (2025)). Specifically, oligonucleotides with separately synthesized cap structures are covalently bonded to some of the nucleotides constituting the circular RNA by enzymatic or chemical ligation (e.g., click chemistry or ligase linkage). This positions the cap structure at a branched position from the circular skeleton, enabling recognition by eukaryotic translation initiation factors (e.g., eIF4E).

[0053] As a second method, circular RNA may form a non-covalent complex with an oligonucleotide (guide oligo) that has a cap structure (see: Fukuchi, K., Nakashima, Y., Abe, N. et al. Internal cap-initiated translation for efficient protein production from circular mRNA. Nat Biotechnol (2025)). In this case, the end of an antisense oligonucleotide having a sequence complementary to a portion of the circular RNA is pre-modified with a cap structure, and when this oligonucleotide hybridizes with the circular RNA, a functional cap structure is presented on the circular RNA.

[0054] As a third approach, circular RNA may contain a cap structure or a mimic thereof within its cyclization junction site or linker structure (see: Wasinska-Kalwa, M., Mamot, A., Czubak, K. et al. Chemical circularization of in vitro transcribed RNA for exploring circular mRNA design. Nat Commun 16, 6455 (2025)). For example, when cyclizing linear RNA by chemically ligating the 5' and 3' ends (e.g., by a reductive amination reaction utilizing ethylenediamine modification at the 5' end and oxidative cleavage at the 3' end), a modified base structure such as m7G can be incorporated into the ligation site to provide a cap function within the circular structure (endocyclic). These methods enable cap-dependent protein translation, independent of the IRES (internal ribosome entry site).

[0055] Circular RNA (circRNA) may further comprise structures containing a sequence of adenine nucleotides (polyA sequences) or their functional equivalents to improve translation efficiency or stability. While naturally occurring circular RNA typically lacks a 3' end and therefore does not have a polyA tail structure, it is possible to confer polyA-binding protein (PABP) recruitment ability to the circular RNA by chemically or enzymatically linking a polyA-containing structure to the circular backbone.

[0056] As a specific method of conferring poly(A) sequences, a technique of linking chemically synthesized oligonucleotides to linear RNA (see: Chen, H., Liu, D., Guo, J. et al. Branched chemically modified poly(A) tails enhance the translation capacity of mRNA. Nat Biotechnol 43, 194-203 (2025)) can be applied to circular RNA. For example, a specific position in the circular RNA (such as the 2'-OH group of a nucleotide or a specific amino-modified base) can be used as a branching point, and an oligonucleotide chain containing a separately prepared polyA sequence can be covalently bonded to this point. Click chemistry (e.g., azide-alkyne cycloaddition reaction) or ligation techniques using ligases (Ligation-Enabled mRNA-Oligonucleotide Assembly; LEGO, etc.) can be used for this bonding. As a result, the polyA structure is presented as if it were a side chain branched from the circular skeleton, while maintaining the circular structure.

[0057] Furthermore, the polyA structure to be affixed is not limited to a simple linear polyA sequence, but may also have a branched structure (Branched polyA). As described in Chen, H., Liu, D., Guo, J. et al. Branched chemically modified poly(A) tails enhance the translation capacity of mRNA. Nat Biotechnol 43, 194-203 (2025)., by attaching a structure (dendrimer-like or fork-like shape) in which multiple polyA chains are bundled via a branched linker to a circular RNA, it becomes possible to efficiently accumulate a large number of PABPs at a single binding site. In addition, the ribose and phosphate backbone contained in the polyA sequence may be chemically modified (e.g., 2'-O-methylation, phosphorothioate, etc.) to enhance exonuclease resistance. With such a configuration, the intracellular stability of the circular RNA is improved, and the protein translation efficiency from the circular RNA is dramatically increased through the interaction between PABP and translation initiation factors (e.g., eIF4G). Furthermore, by genetically engineering the placement of a continuous adenine nucleotide sequence within the circularized RNA sequence, a pseudo-polyA tail function can be conferred.

[0058] One specific introduction method involves inserting a predetermined length of adenine sequence (for example, an adenine sequence of 10 bases or more, preferably 30 to 60 bases) into the untranslated region within the circularized region (for example, the spacer region from the 3' end of the coding region to the IRES or splicing receptor site) during the design phase of the circular RNA expression vector or template DNA (see: Shigetoshi Kameda, Hirohisa Ohno, Hirohide Saito, Synthetic circular RNA switches and circuits that control protein expression in mammalian cells, Nucleic Acids Research, Volume 51, Issue 4, 28 February 2023, Page e24, https: / / doi.org / 10.1093 / nar / gkac1252).

[0059] The circular RNA produced by this method automatically retains the polyA sequence internally during the transcription and cyclization (backsplicing, etc.) processes, without undergoing chemical modifications or post-processing ligation. This "internal polyA tract" specifically binds to polyA-binding proteins such as PABPC1 (Poly(A)-binding protein cytoplasmic 1) within the cell.

[0060] The bound PABPC1 then interacts with translation initiation factors (such as eIF4G) also bound to the IRES (internal ribosome entry site) on the circular RNA, promoting the formation of a translation initiation complex. This activates a translation activation mechanism on the circular RNA that mimics the closed-loop structure of linear mRNA, significantly improving protein production efficiency and translation persistence. Therefore, "polyA-conjugated circular RNA" includes not only polyA tails as terminal structures, but also forms in which a functional polyA sequence is incorporated within the circular sequence.

[0061] Self-amplifying RNA (saRNA) molecules may also be self-replicating RNA (saRNA) molecules. In this specification, "self-amplifying RNA" refers to synthetic RNA molecules that possess self-replication ability using replication mechanisms derived from viruses, etc. Typical self-amplifying RNA is based on an alphavirus genome and includes a replication enzyme gene encoding RNA-dependent RNA polymerase (RdRP) and a gene encoding the target protein. Any RNA-dependent RNA polymerase (RdRP) capable of self-replication can be used in self-replication RNA, and does not necessarily have to be alphavirus-derived or viral-derived. This structure allows RNA to self-replicate within the cell, enabling large-scale and sustained expression of the target protein. Self-amplifying RNA has the advantage of achieving long-term gene expression with lower doses compared to conventional mRNA that does not possess self-replication ability. Furthermore, self-amplifying RNA is often used in combination with delivery systems such as lipid nanoparticles (LNPs). This term encompasses self-replicating RNA molecules used in various biomedical applications, including vaccine development, gene therapy, and protein expression systems.

[0062] mRNA In this specification, the term “messenger RNA” (mRNA) means any ribonucleic acid that codes for (at least one) protein (natural, unnatural, or modified polymer of amino acids) and can be translated in vitro, in vivo, in situ, or ex vivo to produce the coded protein. Those skilled in the art will understand that, unless otherwise stated, nucleic acid sequences described in this application may enumerate “T” within representative DNA sequences, but where the sequence corresponds to RNA (e.g., mRNA), “T” is replaced by “U”. Accordingly, any DNA disclosed herein and identified by a specific sequence identification number also discloses an RNA (e.g., mRNA) sequence transcribed from that DNA, in which case each “T” in the DNA sequence is replaced by “U”.

[0063] mRNA can be naturally occurring or unnatural. For example, mRNA may contain one or more modified and / or unnatural components (such as a nucleobase, nucleoside, nucleotide, or linker). mRNA may contain a cap structure, a chain termination nucleoside, a stem-loop, a poly(A) sequence, and / or a polyadenylation signal. mRNA may have a nucleotide sequence that codes for a polypeptide. Polypeptides may be produced by the translation of mRNA, for example, in vivo translation of mRNA in mammalian cells. Traditionally, the basic components of an mRNA molecule include at least a coding region and an untranslated region (UTR). mRNA may also be circular mRNA.

[0064] The RNA molecule has a linear single-stranded structure and may have a 5'-UTR, an open reading frame (ORF) encoding the desired protein, and a 3'-UTR, oriented from 5' to 3' from the 5' end. The 5'-UTR may include a Cap structure at the 5' end of the RNA molecule. The 3'-UTR may include a poly(A) structure at the 3' end of the RNA molecule.

[0065] An open reading frame (ORF) is a continuous sequence of DNA or RNA that begins with a start codon (e.g., methionine (ATG or AUG)) and ends with a stop codon (e.g., TAA, TAG, or TGA, or UAA, UAG, or UGA). ORFs typically code for proteins. An open reading frame is also called a coding region or coding sequence (CDS).

[0066] In this specification, the term "UTR" means an untranslated region, and refers to untranslated regions (5'UTR, and 3'UTR) located upstream (5') and / or downstream (3') of the coding region of a nucleic acid molecule described herein. Typically, these regions are adjacent to the coding region. UTRs may include "regulators." Regulators refer to nucleic acid sequences that can influence the expression of a target protein. Examples include promoters, enhancers, internal ribosome entry sites (IRES), introns, readers, transcription termination signals (such as polyadenylation signals), and poly-U sequences.

[0067] The structure of the 5'UTR is not particularly limited in terms of the number of bases or the sequence, as long as it has a Cap structure at the 5' end and does not have a short single-stranded nucleic acid target domain. For example, the structure of the 5'UTR is 20 bases or more, for example, consisting of 40 to 150 bases, preferably about 40 to 100 bases, and can be a sequence that does not easily take on an RNA structure such as a stem-loop and does not contain a start codon, but is not limited to a specific sequence.

[0068] The UTR contained in an RNA molecule may have a sequence that is not cleaved by natural RNA other than short single-stranded nucleic acids that targets the target sequence of the short single-stranded nucleic acid target domain. The 5' UTR contained in an RNA molecule may have a sequence that does not contain a start codon (ATG).

[0069] When the RNA molecule of the present invention is a circular RNA molecule, all parts other than the coding region are untranslated regions (UTRs). Within the UTR, the portion whose distance from the 5' end of the target coding region is shorter than the distance from the 3' end of the target coding region can be considered as the 5'UTR of the circular RNA, the portion whose distance from the 3' end of the target coding region is shorter than the distance from the 5' end of the target coding region can be considered as the 3'UTR of the circular RNA, and the portion whose distance from the 3' end of the target coding region is the same as the distance from the 5' end of the target coding region can be considered as both the 5'UTR and the 3'UTR of the circular RNA.

[0070] In this specification, "cap" or "cap structure" refers to a modified structure present at the 5' end of eukaryotic messenger RNA (mRNA). A typical cap structure is m7GpppN, in which 7-methylguanosine (m7G) is linked to the first nucleotide of mRNA via a 5'-5' triphosphate bond. This cap structure plays a crucial role in enhancing mRNA stability, protecting it from degradation by exonucleases, and improving translation efficiency through interaction with translation initiation factors. This term includes not only natural cap structures but also artificial cap analogs (such as ARCA: Anti-Reverse Cap Analogue) introduced into synthetic mRNA. Furthermore, cap structures may undergo additional modifications such as methylation, and these modified caps (e.g., Cap0, Cap1, Cap2) are also included in this term.

[0071] Poly-A In this specification, "poly-A" refers to a sequence of adenine (A) nucleotides located at the 3' end of eukaryotic messenger RNA (mRNA). This structure is usually added as a post-transcriptional modification by poly-A synthase. A typical poly-A sequence consists of approximately 50 to 250 adenine residues, but its length varies depending on the species and gene. The poly-A tail plays an important role in increasing mRNA stability, promoting transport to the cytoplasm, and improving translation efficiency. It also contributes to mRNA circularization and the recruitment of translation initiation factors through interaction with poly-A binding proteins (PABPs). This term includes not only naturally occurring poly-A sequences but also artificial poly-A sequences introduced into synthetic mRNA. Furthermore, variants of poly-A sequences with optimized length and composition (e.g., synthetic poly-A sequences, poly-A / poly-U mixed sequences) are also included in this term. There is no particular upper limit to the length of the polyA sequence, but it may be a sequence consisting of A, for example, 50 to 300 bases, preferably 100 to 150 bases.

[0072] Short Single-Stranded Nucleic Acids Short single-stranded nucleic acids are not particularly limited as long as they are single-stranded nucleic acid molecules composed of 200 or fewer nucleotides, and can bind to target sequences contained in the target domain of short single-stranded nucleic acids. Short single-stranded nucleic acids may be short single-stranded RNA or short single-stranded DNA. Short single-stranded RNA (short single-strand RNA, short ssRNA) Short single-stranded RNA are not particularly limited as long as they are RNA molecules composed of 200 or fewer nucleotides that do not code for proteins, and can bind to target sequences contained in the target domain of short single-stranded nucleic acids. The number of nucleotides constituting short single-stranded RNA is 180 or less, 150 or less, 120 or less, 100 or less, 80 or less, 60 or less, 50 or less, 40 or less, 30 or less, or 25 or less, and 8 or more, 10 or more, 12 or more, 14 or more, 15 or more, 16 or more, 17 or more, 18 or more, 19 or more, or 20 or more.

[0073] Short single-stranded RNA can be native or non-native RNA, and examples include ncRNA, small RNA, miRNA, shRNA, and dicer cleavage products of siRNA. Short single-stranded RNA may also be native miRNA. The number of nucleotides constituting the short single-stranded RNA is, for example, 18 to 25, preferably 20 to 25, and most preferably 21 to 23. Furthermore, it is most preferable that the number of nucleotides constituting the short single-stranded RNA be 14 to 15.

[0074] Short single-strand DNA (short ssDNA) is not particularly limited as long as it is a single-stranded DNA molecule composed of 200 or fewer nucleotides, and can bind to target sequences contained in the short single-stranded nucleic acid target domain. The number of nucleotides constituting the short single-strand DNA is 180 or less, 150 or less, 120 or less, 100 or less, 80 or less, 60 or less, 50 or less, 40 or less, 30 or less, or 25 or less, and 8 or more, 10 or more, 12 or more, 14 or more, 15 or more, 16 or more, 17 or more, 18 or more, 19 or more, or 20 or more. The most preferred number of nucleotides constituting the short single-strand DNA is 20 to 50.

[0075] Short single-stranded DNA is artificially designed DNA, and DNA-type ASOs (antisense oligonucleotides) are an example. The number of nucleotides constituting short single-stranded DNA is, for example, 12-50, 20-50, or 12-28. The number of nucleotides constituting the ASO is most preferably 12-28.

[0076] In this specification, the terms "DNA molecule" and "DNA" refer to nucleic acid molecules containing deoxyribose sugars and comprising at least one base selected from the group consisting of adenine (A), guanine (G), cytosine (C), and thymine (T). DNA typically forms a double-stranded structure, but also includes other structural forms such as single-stranded DNA, circular DNA, and branched DNA. It also includes, but is not limited to, naturally occurring DNA, synthetic DNA, modified DNA, and derivatives thereof.

[0077] In this invention, the "stem" structure of a nucleic acid refers to a double-stranded region formed by the pairing of complementary base sequences within a single-stranded nucleic acid molecule. Stem structures typically exist as part of hairpin loops or stem-loop structures and consist of consecutive base pairs (usually around 2 to 20 base pairs). This structure plays an important role in the secondary structure of RNA and DNA, influencing the stability and function of the molecule. Stem structures may consist of perfectly complementary sequences, or they may include some mismatches or base bulges. This term encompasses not only stem structures found in naturally occurring nucleic acid molecules but also stem structures in artificially designed nucleic acid molecules. Stem structures are used as important structural elements in various functional nucleic acid molecules, such as ribozymes, aptamers, siRNAs, and miRNA precursors.

[0078] When artificially designing a stem structure, it can be done by selecting a portion of the nucleic acid sequence and including a complementary base sequence (antisense) within the same nucleic acid molecule. When artificially setting a stem structure in single-stranded RNA, the selected sequence in the 3' to 5' direction and the antisense in the 5' to 3' direction are arranged to be complementary.

[0079] An RNA molecule contains an aptamer domain, and the aptamer domain may contain a stem. The two sequences that form the stem, one at the 5' end and the other at the 3' end, may each independently have 3 to 10 or 4 to 7 nucleotides, with 4 to 7 nucleotides being most preferable. More than 80% of the two sequences may be complementary. When the sequences at the 5' end and 3' end of the aptamer domain are completely mutual or incompletely complementary, and a hydrogen bond is formed between the sequences at the 5' end and 3' end of the aptamer domain when the aptamer domain forms a secondary structure, the sequences at the 5' end and 3' end of the aptamer domain are said to be stem-forming.

[0080] Nucleic Acid Bases As used herein, the terms “nucleic acid base” (or “nucleotide base” or “nitrogen base”) refer to purine or pyrimidine heterocyclic compounds found in nucleic acids, including derivatives or analogues of natural purines and pyrimidines that confer improved properties (e.g., binding affinity, nuclease resistance, chemical stability) to nucleic acids or parts or fragments thereof. Adenine, cytosine, guanine, thymine, and uracil are the most commonly found nucleic acid bases in natural nucleic acids. Other natural, unnatural, and / or synthetic nucleic acid bases known in the art and / or described herein may be incorporated into nucleic acids.

[0081] Nucleoside / Nucleotide As used herein, the term “nucleoside” refers to a compound in which a sugar molecule (e.g., ribose in RNA or deoxyribose in DNA), or a derivative or analog thereof, is covalently bonded to a nucleic acid base (e.g., purine or pyrimidine), or a derivative or analog thereof (also referred to herein as “nucleic acid base”), but lacks an internucleoside linking group (e.g., a phosphate group). As used herein, the term “nucleotide” refers to a nucleoside, or a derivative, analog, or variant thereof, covalently bonded to an internucleoside linking group (e.g., a phosphate group), which confers improved chemical and / or functional properties (e.g., binding affinity, nuclease resistance, chemical stability) to a nucleic acid or a part or fragment thereof.

[0082] Polypeptides: As used herein, the terms “polypeptide” or “polypeptide of interest” refer to polymers of amino acid residues linked by peptide bonds, which can be produced naturally (e.g., by isolation or purification) or synthetically.

[0083] Proteins In this specification, "protein" refers to a macromolecule in which amino acids are linked together in a linear chain by covalent bonds (peptide bonds). Proteins are usually composed of 20 standard amino acids, and their sequence is encoded by genes. Proteins fold in living organisms to form specific three-dimensional structures, and perform various biological functions based on these structures. This term includes not only naturally occurring proteins but also artificial proteins, recombinant proteins, fusion proteins, and modified proteins (phosphorylation, glycosylation, ubiquitination, etc.) produced by genetic engineering or chemical synthesis. Peptides (generally short amino acid sequences of less than 100 amino acid residues) are also included in this term. Proteins play a diverse range of roles in living organisms, including enzymes, structural proteins, signaling molecules, and antibodies.

[0084] miRNA In this specification, miRNA (micro-RNA, miR, microRNA) refers to a single-stranded RNA of about 18 to 25 nucleotides in length that does not code for proteins. The number of nucleotides in miRNA is, for example, 18 to 25, preferably 20 to 25, and most preferably 21 to 23. In this specification, "miRNA" refers to mature miRNA. In this specification, miRNA only needs to be able to form an RNA-induced silencing complex (RISC). Therefore, miRNA that can bind to the miRNA target sequence contained in the miRNA target domain of an RNA molecule is a non-coding RNA (ncRNA) with a nucleotide number of, for example, 18 to 25, preferably 20 to 25, and most preferably 21 to 23. In this sense, the target domain of a short single-stranded nucleic acid can be rephrased as the miRNA target domain and the ncRNA target domain. In this specification, miRNA is given as an example of ncRNA.

[0085] miRNAs are endogenous RNAs present in cells, and in this specification, endogenous RNAs present in cells, such as miRNAs, may be referred to as "natural RNAs." For example, miRNA refers to one of the strands of a double-stranded RNA produced by cleaving pre-miRNA, which is produced by partially cleaving pri-mRNA (a single-stranded RNA transcribed from cellular DNA) with a nuclear enzyme called Drosha, using Dicer. A database containing information on approximately 1,000 miRNAs can be used (e.g., miRBase, http: / / microrna.sanger.ac.uk / sequences / index.shtml). Those skilled in the art can retrieve information on any natural miRNA from this database, and can easily extract natural miRNAs that are universally expressed in cells into which the gene for the target protein is introduced using the method of the present invention. Note that miRNA expression refers to miRNAs in which, in cells into which the RNA according to the present invention is introduced, one of the strands of the double-stranded RNA cleaved by Dicer interacts with a predetermined number of proteins to form an RNA-induced silencing complex (RISC).

[0086] In this specification, artificially designed short single-stranded RNA is referred to as "non-natural short single-stranded RNA." When non-natural short single-stranded RNA is introduced into cells, it is usually administered in the form of a short single-stranded RNA precursor. Examples of short single-stranded RNA precursors include short single-stranded RNA and double-stranded RNA molecules containing a complementary strand of short single-stranded RNA.

[0087] In this specification, artificially designed short single-stranded DNA is referred to as "non-natural short single-stranded DNA." When introducing short single-stranded DNA into cells, it can be administered either in the form of short single-stranded DNA or in the form of a short single-stranded DNA precursor. Examples of short single-stranded DNA precursors include short single-stranded DNA and a double-stranded molecule containing the complementary strand of short single-stranded DNA.

[0088] In this specification, short single-stranded RNA expressed within a specific cell is referred to as "endogenous short single-stranded RNA." In this specification, short single-stranded RNA delivered to a specific cell is referred to as "exogenous short single-stranded RNA." Exogenous short single-stranded RNA can be delivered regardless of whether it is expressed in the target cell or not. Exogenous short single-stranded RNA is preferably non-natural short single-stranded RNA or natural short single-stranded RNA that is not expressed in the target cell.

[0089] Short Single-Stranded RNA Precursors In this specification, "short single-stranded RNA precursor" refers to an intermediate in the process of producing mature microRNA (short single-stranded RNA). When the short single-stranded RNA is miRNA, the miRNA precursor is either an RNA molecule in which the single-stranded RNA forms a hairpin structure, or a double-stranded RNA molecule in which mature miRNA and its complementary sequence are hybridized. Single-stranded RNA miRNA precursors that form a hairpin structure mainly include two forms: pri-miRNA (primary miRNA) and pre-miRNA (precursor miRNA). Double-stranded RNA miRNA precursors mainly include siRNA. This term encompasses not only naturally occurring short single-stranded RNA precursors but also artificially designed or modified short single-stranded RNA precursors.

[0090] When the short single-stranded RNA is a miRNA, the natural pri-miRNA is an early transcript transcribed by RNA polymerase II and has a 5' cap and a 3' poly-A tail. The pri-miRNA is cleaved in the nucleus by the Drosha enzyme complex to become a pre-miRNA of approximately 60-70 nucleotides. After being transported to the cytoplasm, the pre-miRNA is further processed by the Dicer enzyme to finally become a mature miRNA of 18-25 nucleotides. When the short single-stranded RNA precursor is an artificially designed shRNA (short hairpin RNA) or siRNA (small interfering RNA), the single-stranded RNA produced by cleavage or hybridization dissociation of these precursors in the cell becomes the short single-stranded RNA described herein. After binding to the target sequence, it interacts with multiple proteins, similar to miRNAs, to form an RNA-induced silencing complex (RISC) that represses protein translation from the RNA molecule containing the target sequence.

[0091] shRNA In this specification, the term "shRNA" (short hairpin RNA) refers to an artificial single-stranded RNA molecule designed to induce RNA interference (RNAi). shRNA has a structure in which two regions containing complementary nucleotide sequences are linked by an intervening sequence (loop), forming a hairpin structure after transcription. Typically, shRNA is expressed in cells from a DNA vector containing a suitable promoter, and then processed by the Dicer enzyme to become an siRNA (small interfering RNA)-like molecule, which is incorporated into the RNA-induced silencing complex (RISC) to cause degradation or translational repression of target mRNA. This term also includes shRNAs designed to mimic the precursor structures of natural microRNAs (miRNAs), as well as shRNA derivatives with various modifications.

[0092] In this invention, "siRNA" (small interfering RNA) refers to a short double-stranded RNA molecule that induces sequence-specific gene silencing via RNA interference (RNAi). siRNA typically has a length of 21–23 base pairs and a 2-base overhang at the 3' end of each strand. siRNA is synthesized intracellularly or introduced exogenously and incorporated into the RNA-induced silencing complex (RISC). The guide strand of the siRNA incorporated into the RISC recognizes a target mRNA with a complementary sequence, causing cleavage or translational repression. This term also includes variants such as chemically modified siRNA, asymmetric siRNA, and Dicer substrate siRNA.

[0093] A short single-stranded nucleic acid target domain refers to a domain containing a sequence that can specifically bind to the short single-stranded nucleic acid. A short single-stranded nucleic acid target domain may also consist solely of a sequence that can specifically bind to the short single-stranded RNA. When the short single-stranded nucleic acid is short single-stranded RNA, the short single-stranded nucleic acid target domain can be rephrased as a short single-stranded RNA target domain. When the short single-stranded nucleic acid is short single-stranded DNA, the short single-stranded nucleic acid target domain can be rephrased as a short single-stranded DNA target domain. In this specification, the "target sequence" in a short single-stranded nucleic acid target domain is used interchangeably with the "binding sequence" in a short single-stranded nucleic acid target domain.

[0094] The short single-stranded RNA target sequence is preferably, for example, a sequence complementary to short single-stranded RNA universally expressed in cells. Alternatively, the short single-stranded RNA target sequence may have a mismatch with a perfectly complementary sequence, as long as it can be recognized by the short single-stranded RNA. The mismatch from the perfectly complementary sequence of the short single-stranded RNA only needs to be a mismatch that the short single-stranded RNA can normally recognize in the desired cell, and it is considered acceptable for a mismatch of about 40-50% to function within the cell in vivo. Such mismatches are not particularly limited, but examples include mismatches of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 bases, or mismatches of 1%, 5%, 10%, 20%, 30%, or 40% of the total recognition sequence. Furthermore, the target sequence may contain numerous discrepancies, particularly in the region other than the seed region, such as the short single-stranded RNA target sequence of the mRNA present in cells, that is, in the 5' region of the target sequence, which corresponds to approximately 16 bases on the 3' side of the short single-stranded RNA. This region may contain discrepancies of 1 to 7 bases. The seed region may contain no discrepancies, or it may contain discrepancies of 1, 2, or 3 bases.

[0095] Seed matching plays a crucial role in the binding of short single-stranded RNA (STRNA) to its target sequence. The seed region generally refers to the 2nd to 8th nucleotides from the 5' end of the STRNA, and binds complementaryly to the 2nd to 8th nucleotides from the 3' end of the STRNA target sequence. This complementarity does not need to be perfect; a continuous complementary binding of 6-7 base pairs is usually sufficient. The principle of seed matching is that complementary binding in this short region is essential for the initial recognition and stabilization of the binding between the STRNA and its target sequence. Once the binding in the seed region is established, it acts as an anchor, promoting further interaction between the entire STRNA and its target sequence. This mechanism enables relatively short STRNAs to recognize and control a variety of target sequences.

[0096] Expression As used herein, the term "expression" refers to RNA expression, and therefore to one or more of the following events relating to a nucleic acid sequence: (1) generation of an RNA template from a DNA sequence (e.g., by transcription); (2) processing of an RNA transcript (e.g., by 5′ cap formation and / or 3′ end processing); (3) translation of RNA into a polypeptide or protein; and (4) post-translational modification of a polypeptide or protein. As used herein, the term "expression" refers to protein expression, and therefore to translation of RNA into a polypeptide or protein.

[0097] In this specification, "aptamer" refers to a structure consisting of single-stranded nucleic acids that have been artificially selected to have high affinity and specificity for a particular target molecule. Aptamers are composed of DNA, RNA, or modified nucleic acids and are typically 20 to 100 nucleotides long. The three-dimensional structure of an aptamer plays a crucial role in binding to the target molecule and may include structural elements such as stem-loops, G-quadraplexes, and pseudoknots. Aptamers are designed for a wide range of targets, including proteins, small molecules, cells, and viruses, and have a broad range of applications in diagnostics, therapeutics, biosensors, and analytical technologies. This term encompasses aptamers obtained by the SELEX (Systematic Evolution of Ligands by Exponential Enrichment) method and other selection methods, as well as their modifications.

[0098] The aptamers contained in the RNA molecule of the present invention have a stable stem structure of about 2 to 20 base pairs, preferably 3 to 10 base pairs, at their root (5' or 3' end). This stem structure improves the overall structural stability of the aptamer and enables the proper presentation of the target binding site.

[0099] Transfection In this specification, the term “transfection” refers to the introduction of nucleic acid molecules, such as DNA or RNA (e.g., mRNA) molecules, into cells, preferably eukaryotic cells. With respect to the present invention, the term “transfection” encompasses all methods known to those skilled in the art for introducing nucleic acid molecules into cells, preferably eukaryotic cells (such as mammalian cells). Such methods include, for example, electroporation, lipofection (e.g., lipofection based on cationic lipids and / or liposomes), calcium phosphate precipitation, nanoparticle-based transfection, virus-based transfection, or cationic polymer-based transfection (e.g., DEAE-dextran or polyethyleneimine). Preferably, the introduction is carried out nonvirally.

[0100] Vectors As used herein, the term "vector" refers to a nucleic acid molecule that can amplify another nucleic acid to which it is linked. This term includes vectors as self-replicating nucleic acid structures, and vectors that are incorporated into the genome of a host cell into which they are introduced. Some vectors can result in the expression of the nucleic acid to which they are operationally linked. Such vectors are also referred to herein as "expression vectors." Vectors can be introduced into host cells by methods such as viruses or electroporation, but vector introduction is not limited to in vitro; it is also possible to introduce vectors directly into living organisms.

[0101] Modified Nucleotides In this specification, “modified nucleotide” refers to an artificial nucleotide derivative that has been chemically modified from a natural nucleotide (deoxyribonucleotide or ribonucleotide). Modifications are made to the base portion, sugar portion (ribose or deoxyribose), phosphate portion, or a combination thereof of the nucleotide. Examples of modifications include, but are not limited to, methylation, fluorination, 2'-O-methylation, 2'-F substitution, locked nucleic acid (LNA), phosphorothioate linkage, and peptide nucleic acid (PNA). Modified nucleotides are designed to improve the stability of nucleic acid molecules, enhance nuclease resistance, improve cell membrane permeability, increase target binding affinity, or confer specific functions. This term encompasses not only nucleotides with a single modification but also nucleotides with a combination of multiple modifications.

[0102] RNA molecules may contain modified nucleotides. When RNA molecules in this specification contain modified nucleotides, it is preferable that they contain artificial nucleotide derivatives obtained by chemically modifying ribonucleotides.

[0103] The DNA molecule may contain modified nucleotides. If the DNA molecule in this specification contains modified nucleotides, it is preferable that it contains artificial nucleotide derivatives obtained by chemically modifying deoxyribonucleotides.

[0104] Modified nucleotides may include nucleotide base modifications and may undergo further modifications. Examples of nucleoside modifications include 6-azacytidine, 2-thiocytidine, α-thiocytidine, pseudoisocytidine, 5-aminoallyluridine, 5-iodouridine, N1-methylpsoiduridine, 5,6-dihydrouridine, α-thiouridine, 4-thiouridine, 6-azauridine, 5-hydroxyuridine, deoxythymidine, 5-methyluridine, pyrrolocytidine, inosine, and α-thio- Nucleotide base modifications can be selected from anosine, 6-methyl-guanosine, 5-methyl-cytidine, 8-oxo-guanosine, 7-deaza-guanosine, N1-methyl-adenosine, 2-amino-6-chloropurine, N6-methyl-2-aminopurine, pseudo-isocytidine, 6-chloropurine, N6-methyl-adenosine, α-thio-adenosine, 8-azido-(BR>A)denosine, and 7-deaza-adenosine.

[0105] Further examples of nucleotide base modifications include pyridine-4-onribonucleoside, 5-azauridine, 2-thio-5-azauridine, 2-thiouridine, 4-thiopsoiduridine, 2-thiopsoiduridine, 5-hydroxyuridine, 3-methyluridine, 5-carboxymethyluridine, 1-carboxymethylpsoiduridine, 5-propynyluridine, 1-propynylpsoiduridine, 5-taurinomethyluridine, 1-taurinomethylpsoiduridine, 5-taurinomethyl-2-thiouridine, and 1-taurinomethyl Examples include -4-thiouridine, 5-methyluridine, 1-methylpsoiduridine, 4-thio-1-methylpsoiduridine, 2-thio-1-methylpsoiduridine, 1-methyl-1-deazalpsoiduridine, 2-thio-1-methyl-1-deazalpsoiduridine, dihydrouridine, dihydropsoiduridine, 2-thio-dihydrouridine, 2-thio-dihydropsoiduridine, 2-methoxyuridine, 2-methoxy-4-thiouridine, 4-methoxypsoiduridine, and 4-methoxy-2-thiopsoiduridine.

[0106] Further examples of nucleotide base modifications include 5-azacytidine, pseudoisocytidine, 3-methylcytidine, N4-acetylcytidine, 5-formylcytidine, N4-methylcytidine, 5-hydroxymethylcytidine, 1-methylpsoidisocytidine, pyrrolocytidine, pyrrolocytidine, 2-thiocytidine, 2-thio-5-methylcytidine, 4-thiopsoidisocytidine, and 4-thio-1-methylps Examples include soidisocytidine, 4-thio-1-methyl-1-deazal-psoidisocytidine, 1-methyl-1-deazal-psoidisocytidine, zebralin, 5-aza-zebralin, 5-methyl-zebralin, 5-aza-2-thio-zebralin, 2-thio-zebralin, 2-methoxycytidine, 2-methoxy-5-methylcytidine, 4-methoxy-psoidisocytidine, and 4-methoxy-1-methyl-psoidisocytidine.

[0107] Further examples of nucleotide base modifications include 2-aminopurine, 2,6-diaminopurine, 7-deaza-adenine, 7-deaza-8-aza-adenine, 7-deaza-2-aminopurine, 7-deaza-8-aza-2-aminopurine, 7-deaza-2,6-diaminopurine, 7-deaza-8-aza-2,6-diaminopurine, 1-methyladenosine, N6-methyladenosine, N6-isopentenyladenosine, N6- Examples include (cis-hydroxyisopentenyl)adenosine, 2-methylthio-N6-(cis-hydroxyisopentenyl)adenosine, N6-glycinylcarbamoyladenosine, N6-threonylcarbamoyladenosine, 2-methylthio-N6-threonylcarbamoyladenosine, N6,N6-dimethyladenosine, 7-methyladenine, 2-methylthioadenine, and 2-methoxyadenine.

[0108] Further examples of nucleotide base modifications include inosine, 1-methylinosine, waiosine, waibutosine, 7-deaza-guanosine, 7-deaza-8-aza-guanosine, 6-thio-guanosine, 6-thio-7-deaza-8-aza-guanosine, 7-methyl-guanosine, 6-thio-7-methyl-guanosine, 7-methylinosine, 6-methoxy-guanosine, 1-methylguanosine, N2-methylguanosine, N2,N2-dimethylguanosine, 8-oxo-guanosine, 7-methyl-8-oxo-guanosine, 1-methyl-6-thioguanosine, N2-methyl-6-thioguanosine, and N2,N2-dimethyl-6-thioguanosine.

[0109] The modified nucleotide may be a modified nucleotide that includes modifications in the phosphate backbone, and may be further modified. The phosphate group of the backbone may be modified by substituting one or more oxygen atoms with other substituents. The modified nucleotide may include total substitution of the unmodified phosphate portion with a modified phosphate salt.

[0110] Examples of modified phosphate groups include phosphorothioates, phosphoroselenates, boranophosphates, boranophosphate esters, hydrogen phosphonates, phosphoramidates, alkyl or aryl phosphonates, and phosphotriesters. In phosphorothioates, both non-linking oxygen atoms are replaced with sulfur. Phosphorate linkers can also be modified by substituting the linking oxygen atoms with nitrogen (bridged phosphoramidate), sulfur (bridged phosphorothioate), and carbon (bridged methylene-phosphonate).

[0111] Examples of modified phosphate groups include the incorporation of nonionic phosphate analogs such as alkyl and aryl phosphonates, in which the charged phosphonate oxygen is substituted with an alkyl or aryl group, or the charged oxygen residue is substituted with a phosphodiester or alkylphosphotriester present in an alkylated form. Such backbone modifications typically include modifications from the group consisting of methyl phosphonates, phosphoramidates, and phosphorothioates (e.g., cytidine-5'-O-(1-thiophosphate)).

[0112] Modified nucleotides may include sugar modifications and may undergo further modifications. In this specification, the term "sugar modification" means chemical modification of the sugar moiety of a nucleotide. The 2'-hydroxyl group (OH) may be converted, for example, by deoxy conversion, alkoxy or aryloxy (-OR, e.g., R=H, alkyl, cycloalkyl, aryl, aralkyl, heteroaryl or sugar); polyethylene glycol (PEG), -O(CH) ２ CH ２ O) ｎ CH ２ CH ２ OR; locked nucleic acids (LNA) in which a 2'-hydroxyl group is linked to the 4'-carbon of the same ribose sugar, for example by a methylene crosslink; and amino groups (-O-amino, where the amino group (e.g., NRR) may be alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino or diheteroarylamino, ethylenediamine, polyamino) or aminoalkoxys.

[0113] Examples of deoxy modification include hydrogen and amino acids (e.g., NH₄). ２ (containing alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino, diheteroarylamino, or amino acids); or the amino group may be bonded to the sugar via a linker, where the linker contains one or more atoms C, N, and O.

[0114] Examples of modified sugars include those containing one or more carbon atoms with stereochemical configurations opposite to those of the corresponding carbon atoms in ribose. Therefore, sugar-modified artificial nucleic acid (RNA) molecules may contain, for example, nucleotides containing arabinose as the sugar.

[0115] Modified nucleotides can be synthesized by any useful method, such as chemically, enzymatically, or recombinantly, to contain one or more modifications or non-natural nucleosides. Nucleic acids may contain regions of linked nucleosides. Such regions may have variable backbone linkages. The linkage may be a standard phosphate diester bond, in which case the nucleic acid will contain a region of nucleotides.

[0116] Base pairing of modified nucleotides includes not only standard adenosine-thymine, adenosine-uracil, or guanosine-cytosine base pairs, but also base pairs formed between nucleotides containing non-standard or modified bases and / or between modified nucleotides. Here, the arrangement of hydrogen bond donors and hydrogen bond acceptors enables hydrogen bonding between non-standard and standard bases, or between two complementary non-standard base structures. This is observed in nucleic acids having at least one chemical modification. An example of such a non-standard base pairing is the base pairing of the modified nucleotide inosine with adenine, cytosine, or uracil. Any combination of base / sugar or linker can be incorporated into the nucleic acids of this disclosure.

[0117] Modified nucleobases in nucleic acids (e.g., nucleic acids such as RNA, nucleic acids such as mRNA) may include N1-methyl-pseudridine (m1ψ), 1-ethyl-pseudridine (e1ψ), 5-methoxy-uridine (mo5U), 5-methyl-cytidine (m5C), and / or pseudouridine (ψ). Modified nucleobases in nucleic acids (e.g., nucleic acids such as RNA, nucleic acids such as mRNA) may also include 5-methoxymethyluridine, 5-methylthiouridine, 1-methoxymethylpseudridine, 5-methylcytidine, and / or 5-methoxycytidine. Polyribonucleotides may contain a combination of at least two (e.g., 2, 3, 4 or more) of the above modified nucleobases, including but not limited to chemical modifications.

[0118] Carrier In this specification, "carrier" refers to a medium or carrier used to deliver nucleic acid molecules to target cells or tissues. Carriers have the function of protecting nucleic acids from physical and chemical degradation, improving cell membrane permeability, and enabling efficient delivery to the target site. Examples of carriers include, but are not limited to, lipid nanoparticles (LNPs), liposomes, polymer nanoparticles, dendrimers, exosomes, viral vectors, peptide carriers, and inorganic nanoparticles. Carriers are appropriately selected or designed depending on the type of nucleic acid (DNA, RNA, oligonucleotides, etc.), the characteristics of the target cell or tissue, and the route of administration. This term encompasses not only carriers consisting of a single component but also complex carrier systems combining multiple components. Carriers play a crucial role in various nucleic acid-based therapies and diagnostics, such as nucleic acid drugs, gene therapy, RNA interference therapy, and vaccines.

[0119] As used herein, "liposome" refers to a structure comprising a lipid-containing membrane enclosing an aqueous interior. Liposomes may have one or more lipid membranes. Liposomes include single-layer liposomes (also known in the art as monolayer liposomes) and multilayer liposomes (also known in the art as multilayer liposomes).

[0120] Liposomes may be used as carriers for the delivery of polynucleotides (RNA or DNA molecules). Known liposomes such as multilayered vesicles (MLVs) containing aqueous compartments separated by concentric bilayers, small diameter unicellular vesicles (SUVs), and large diameter monolayered vesicles (LUVs) may be used. The diameter of the liposome may be 1 μm or less, 700 nm or less, 500 nm or less, 300 nm or less, 150 nm or less, 120 nm or less, 100 nm or less, or 50 nm or less, and may be 10 nm or more, 50 nm or more, 80 nm or more, for example, 50 to 500 nm. The diameter of the liposome is preferably 10 to 200 nm, most preferably 80 to 120 nm when it is desired to increase blood retention, most preferably 100 to 200 nm when delivered to the liver, and most preferably 30 to 50 nm when it is desired to penetrate fibrotic tissue.

[0121] Lipid Nanoparticles In this specification, "lipid nanoparticles" (LNPs) refer to nanoscale particulate structures primarily composed of lipids, serving as carrier systems for the efficient delivery of nucleic acids and other therapeutic agents. LNPs typically have a diameter in the range of 20–200 nm and are composed of multiple lipid components, such as cationic lipids, neutral lipids, cholesterol, and PEGylated lipids. LNPs have the function of encapsulating nucleic acids (e.g., mRNA, siRNA, plasmid DNA) or small molecule drugs and protecting them from physical and chemical degradation. LNPs also facilitate cellular uptake and enable escape from endosomes, thereby achieving efficient delivery of encapsulated drugs into the cytoplasm. The composition, size, and surface properties of LNPs are optimized according to the target cells or tissues, the characteristics of the encapsulated drug, and the administration route. This term refers to nanoparticles with more complex lipid compositions and structures than conventional liposomes, and is widely used, particularly in the delivery of nucleic acid drugs.

[0122] Lipid nanoparticles may be used as carriers for the delivery of polynucleotides (RNA molecules or DNA molecules). The lipid nanoparticles may include lipids selected from the group consisting of DLin-DMA, DLin-K-DMA, 98N12-5, C12-200, DLin-MC3-DMA, DLin-KC2-DMA, DODMA, PLGA, PEG, PEG-DMG, PEG-modified lipids, amino alcohol lipids, KL22, and combinations thereof.

[0123] Lipid nanoparticles may have diameters of 10 nm or more, 20 nm or more, or 30 nm or more, and 500 nm or less, 300 nm or less, 200 nm or less, 100 nm or less, 90 nm or less, 80 nm or less, or 70 nm or less. Lipid nanoparticles may have diameters of, for example, 10 to 500 nm, preferably 10 to 200 nm, more preferably 10 to 100 nm, and most preferably 60 to 100 nm. When delivered to the lungs or immune cells, the most suitable diameter for lipid nanoparticles is 150 to 200 nm. When tissue penetration is to be enhanced, the most suitable diameter for lipid nanoparticles is 10 to 50 nm.

[0124] Polymer Nanoparticles In this specification, "polymer nanoparticles" refer to nanoscale particulate structures primarily composed of synthetic or natural polymers, serving as carrier systems for the efficient delivery of drugs and biomolecules. Polymer nanoparticles typically have a diameter in the range of 10 to 1000 nm and are composed of biodegradable or non-biodegradable polymers. Examples of polymers used include, but are not limited to, polylactic acid-glycolic acid copolymers (PLGA), polyethylene glycol (PEG), chitosan, and sodium alginate. Polymer nanoparticles have the function of encapsulating or adsorbing nucleic acids, proteins, peptides, and small molecule drugs, protecting them from physical and chemical degradation. Furthermore, polymer nanoparticles can be given target-directivity through surface modification of the particles, contributing to controlled drug release and improved biocompatibility. The composition, size, and surface properties of polymer nanoparticles are optimized according to the target cells or tissues, the properties of the drug to be encapsulated, and the administration route. This term encompasses various forms of polymer-based nanostructures, including nanospheres, nanocapsules, micelles, and dendrimers.

[0125] Nanoparticles As used herein, "nanoparticles" refers to particles that have arbitrary structural features on a scale of less than 1000 nm and exhibit novel properties compared to bulk samples of the same material. Typically, nanoparticles have arbitrary structural features on a scale of less than 500 nm, less than 200 nm, or about 100 nm. Generally, nanoparticles have arbitrary structural features on a scale of about 50 nm to about 500 nm, preferably about 50 nm to about 200 nm, and most preferably about 70 nm to about 120 nm.

[0126] Nanoparticles may be particles having one or more dimensions on the order of approximately 1 to 1000 nm. Nanoparticles may be particles having one or more dimensions on the order of approximately 10 to 500 nm. Nanoparticles may be particles having one or more dimensions on the order of approximately 50 to 200 nm. In the case of spherical nanoparticles, for example, the diameter will be between approximately 50 to 100 nm or 70 to 120 nm.

[0127] Nanoparticles, in terms of their transport and properties, almost always behave as a single unit. Novel properties that distinguish nanoparticles from their corresponding bulk materials typically manifest on a size scale of less than 1000 nm, preferably around 100 nm, although nanoparticles can also be larger (e.g., elongated particles, tubular particles, etc.). While the sizes of most molecules fit within the above outline, individual molecules are not usually referred to as nanoparticles.

[0128] Where used herein, the term "viral particle" has the general meaning in the art and refers to a fully or partially assembled viral capsid or an envelope formed by a lipid bilayer. Viral particles may or may not contain a viral genome. Therefore, this term may include virus-like particles (VLPs). Viral particles have a diameter of approximately 20–150 nm and also possess nanometer material properties such as a large surface area, surface-accessible amino acids with reactive groups (such as lysine and glutamate residues), an irregular spatial structure, and excellent biocompatibility. Therefore, viral particles have great potential as a delivery system for carrying various cargoes.

[0129] Virus-like particles: As used herein, the terms “virus-like particles” or “VLPs” refer to structures that resemble virus particles but do not contain a viral genome, are incapable of replication, and are non-pathogenic. Virus-like particles typically contain at least one viral or non-viral structural protein. A virus-like particle may contain only one structural protein, and may not contain other non-structural components of a virus. Virus-like particles, while lacking genetic material and eliminating the potential for replication, can spontaneously self-assemble in vitro through their structural proteins under appropriate conditions. Virus-like particles have a diameter of approximately 20–150 nm and also possess nanometer material properties such as a large surface area, surface-accessible amino acids with reactive regions (such as lysine and glutamate residues), irregular spatial structures, and excellent biocompatibility. Therefore, virus-like particles have great potential as delivery systems for carrying various cargoes.

[0130] Enveloped Virus Particles In this specification, virus particles or virus-like particles may include “enveloped virus particles” or “enveloped virus-like particles.” Enveloped virus particles refer to virus particles surrounded by a lipid bilayer envelope derived from the cell membrane, and enveloped virus-like particles refer to virus-like particles surrounded by a lipid bilayer envelope derived from the cell membrane. “Cell membrane-derived lipid bilayer envelope” refers to a lipid bilayer derived from the cell membrane of the host cell from which the virus particle was released. This envelope partially or completely encloses the virus particle or virus-like particle. The virus particle or virus-like particle may be completely (or substantially completely) enclosed within the envelope. The lipid bilayer may have a polymeric composition corresponding to the composition of the host cell membrane. The lipid bilayer may contain lipids, proteins, and hydrocarbons found in the host cell membrane in similar proportions to those in the host cell membrane. Such macromolecules may include transmembrane receptors and channels (receptor kinases, ion channels, etc.), cytoskeletal proteins (actin, etc.), lipids or protein-binding hydrocarbons, phospholipids (phosphatidylcholine, phosphatidylserine, phosphatidylethanolamine, etc.), and cholesterol.

[0131] Extracellular Vesicles: As used herein, the term “extracellular vesicle” has the general meaning in the art. Extracellular vesicles are known to vary in size, origin, and markers, and include, for example, exosomes, Arrestin domain-containing protein 1-mediated microvesicles, small ectosomes, microvesicles, apoptotic bodies / vesicles, migrasomes, and large oncosomes (see Trends in Cell Biology, August 2023, Vol. 33, No. 8, 667-681), and cell-derived vesicles (see http: / / www.mdimune.com / en / m61.php; US 2018 / 0296483 A1).

[0132] Envelope Proteins: As used herein, the term "envelope protein" refers to proteins encoded by the viral genome and associated with the viral envelope in typical enveloped viruses, or envelope proteins of endogenous retroviruses. Envelope proteins can, for example, specifically interact with cellular receptor proteins to facilitate the attachment of viral particles or virus-like particles to cells. Examples of envelope proteins include, but are not limited to, glycoproteins.

[0133] Administration As used herein, “administration” means a method of delivering a composition to a subject or patient. The method of administration may be selected for targeted delivery to a specific area or system of the body (e.g., specific delivery). For example, administration may be performed by any of the following methods: parenteral (e.g., subcutaneous, intradermal, intravenous, intraperitoneal, intramuscular, intra-articular, intra-arterial, intra-synovial, intrasternal, intrathecal, intrafocal, or intracranial injection, and appropriate infusion techniques), oral, transdermal or intradermal, interdermal, rectal, vaginal, topical (e.g., by powder, ointment, cream, gel, lotion, and / or eye drops), mucous membrane, intranasal, buccal, intestinal, intravitreous, intratumoral, sublingual, intranasal; by intratracheal infusion, intrabronchial infusion, and / or inhalation; as oral spray and / or powder, nasal spray, and / or aerosol; and via portal vein catheter. Preferred methods of administration are intravenous or subcutaneous.

[0134] Delivery As used herein, the term “delivery” means providing an entity to a destination. For example, delivering one or more RNA or DNA molecules of this disclosure to a subject may include administering a composition (e.g., lipid nanoparticles (LNPs) comprising one or more polynucleotides) to a subject (e.g., by intravenous, intramuscular, intradermal, pulmonary, or subcutaneous routes). Administering a composition (e.g., LNPs) to a mammal or mammalian cells may include bringing one or more cells into contact with the composition.

[0135] Contact As used herein, the term “contact” means establishing a physical connection between two or more entities. For example, contacting a cell with an RNA or lipid nanoparticle composition means causing the cell and the mRNA or lipid nanoparticles to share a physical connection. Methods for contacting cells with external entities are well known in the field of biology, whether in vivo, in vitro, or ex vivo. The step of contacting mammalian cells with a composition (e.g., nanoparticles, or the pharmaceutical composition of this disclosure) may be carried out in vivo.

[0136] For example, when a lipid nanoparticle composition is brought into contact with cells (e.g., mammalian cells), this can be done by an appropriate route of administration (e.g., parenteral administration, intravenous administration, intramuscular administration, intradermal administration, and subcutaneous administration) even if the cells are located within an organism (e.g., a mammal). In the case of cells present in vitro, contact between the composition (e.g., lipid nanoparticles) and the cells can be done, for example, by adding the composition to the cell culture medium, which may involve or result in transfection. Furthermore, a single nanoparticle composition may come into contact with multiple cells.

[0137] Specific Delivery As used herein, the terms “specific delivery,” “deliver specifically,” or “delivered specifically” mean that the amount of the disclosed polynucleotide delivered by a delivery agent (e.g., nanoparticles) to target cells of interest (e.g., mammalian target cells) is greater than that delivered to non-target cells (e.g., non-target cells) (e.g., at least 10% more, at least 20% more, at least 30% more, at least 40% more, at least 50% more, at least 1x more, at least 1.5x more, at least 2x more, at least 3x more, at least 4x more, at least 5x more, at least 6x more, at least 7x more, at least 8x more, at least 9x more, at least 10x more). It is most preferable that the amount delivered to target cells of interest is at least twice the amount delivered to non-target cells.

[0138] Encapsulation: As used herein, the term “encapsulation” means to include, surround, or enclose a compound, polynucleotide (e.g., mRNA), or other composition, which may be fully encapsulated, partially encapsulated, or substantially encapsulated. For example, mRNA of this disclosure may be encapsulated in lipid nanoparticles, e.g., liposomes.

[0139] Encapsulation Efficiency As used herein, “encapsulation efficiency” refers to the amount of polynucleotides that become part of the nanoparticle composition relative to the initial total amount of polynucleotides used in the preparation of the nanoparticle composition. For example, if 97 mg of the 100 mg of polynucleotides initially provided in the preparation of the nanoparticle composition are encapsulated, the encapsulation efficiency is expressed as 97%. As used herein, “encapsulation” may refer to complete, substantial, or partial inclusion, containment, surrounding, or encapsulation.

[0140] RNA molecules are provided herein, comprising a sequence encoding a target protein, an aptamer domain capable of binding to a target protein different from the target protein, and a short single-stranded nucleic acid target domain containing a target sequence to which a short single-stranded nucleic acid can be bound. The aptamer domain may contain an aptamer sequence, which can be prepared according to the description herein or by known methods.

[0141] An RNA molecule containing a sequence encoding the protein of interest (the protein to be expressed) may function as mRNA expressing the protein of interest in vivo. The protein of interest may have an effective effect for use in treating, preventing, or diagnosing a target condition or disease when expressed in the target organism. The protein of interest may be an antigen that activates an immune response, an antigen-binding polypeptide (e.g., an antibody or a fragment thereof, a polypeptide containing the antigen-binding site of an antibody such as a single-chain antibody), a transcription activator or repressor of a target gene, a reporter protein, an enzyme, a receptor, or a ligand.

[0142] There is no size limit to the RNA molecule containing the sequence encoding the target protein, as long as it does not exceed the capacity of the carrier for delivery. For example, nanoparticles such as LNPs can typically encapsulate RNA molecules with a sequence length of 10 kb (10,000 bases) or more, and can also encapsulate self-amplified RNA with a sequence length of approximately 30 kb (30,000 bases).

[0143] The target sequence contained in the short single-stranded nucleic acid binding domain is capable of binding to short single-stranded nucleic acids, and the binding of the short single-stranded nucleic acid to the target sequence inhibits the translation of the target protein encoded by the RNA molecule. The target sequence and the short single-stranded nucleic acid are not particularly limited, as long as the binding of the short single-stranded nucleic acid to the target sequence inhibits the translation of the target protein.

[0144] Whether the translation of a target protein encoded by an RNA molecule containing a short single-stranded nucleic acid binding domain is inhibited by the binding of the short single-stranded nucleic acid can be measured by comparing the expression levels of the target protein in cells containing the short single-stranded nucleic acid into which the RNA molecule has been introduced with cells not containing the short single-stranded nucleic acid into which the RNA molecule has been introduced. If the expression level of the target protein in cells containing the short single-stranded nucleic acid into which the RNA molecule containing a short single-stranded nucleic acid binding domain has been introduced is 90% or less, 80% or less, 70% or less, 60% or less, 50% or less, 40% or less, 30% or less, 20% or less, 10% or less, 5% or less, 4% or less, 3% or less, 2% or less, or 1% or less of the expression level of the target protein in cells not containing the short single-stranded nucleic acid into which the RNA molecule has been introduced, it means that the translation of the target protein encoded by the RNA molecule is inhibited by the binding of the short single-stranded nucleic acid. If the expression level of the target protein in cells containing short single-stranded nucleic acids into which an RNA molecule containing a short single-stranded nucleic acid binding domain has been introduced is below the detection limit, it means that the translation of the target protein encoded by the RNA molecule is inhibited by the binding of the short single-stranded nucleic acid. When measuring whether the translation of the target protein encoded by an RNA molecule containing a short single-stranded nucleic acid binding domain is inhibited by the binding of the short single-stranded nucleic acid, it is necessary that the cells used do not contain the target protein to which the aptamer domain contained in the RNA molecule binds.

[0145] The quantification of intracellular protein expression levels can be performed using biological or chemical measurement techniques known in the art. While the method of protein quantification is not particularly limited, immunological methods utilizing specific affinity for target proteins, or comprehensive analysis methods based on mass spectrometry, are preferably used.

[0146] Immunological methods include Western blotting, enzyme-linked immunosorbent assay (ELISA), dot blotting, immunoprecipitation, immunohistochemical staining, or flow cytometry (FACS) using antibodies that specifically bind to the target protein. Furthermore, if the target protein is expressed through exogenous gene transfer, detection or quantification may be performed using antibodies against a tag sequence attached to the protein (e.g., FLAG tag, His tag, HA tag, Myc tag, or fluorescent protein such as GFP). These methods make it possible to measure the presence of a specific protein and its relative or absolute quantity.

[0147] Furthermore, if more comprehensive and sensitive quantification is required, or if obtaining specific antibodies is difficult, quantitative analysis using mass spectrometry (MS) can be employed. Specifically, proteins contained in cell extracts can be digested into peptide fragments using enzymes such as trypsin, and then shotgun proteomics analysis can be performed using LC-MS / MS, which combines liquid chromatography and tandem mass spectrometry.

[0148] The short single-stranded nucleic acid may bind to a target sequence, thereby inducing the degradation of the RNA molecule of the present invention. The short single-stranded nucleic acid bound to the target sequence may interact with multiple proteins to form an RNA-induced silencing complex (RISC), thereby inhibiting the translation of the target protein from the RNA molecule of the present invention. The short single-stranded nucleic acid bound to the target sequence may interact with RNaseH, thereby inhibiting the translation of the target protein from the RNA molecule of the present invention.

[0149] The target sequence may have a sequence to which a short single-stranded RNA ubiquitously expressed within the tissue to which the RNA molecule is delivered can bind. This short single-stranded RNA target sequence may be able to bind to a short single-stranded RNA having any one sequence from sequence numbers 125 to 207. The short single-stranded RNA target sequence may be a sequence that is completely complementary to any one sequence from sequence numbers 125 to 207. The target sequence may have a sequence that is completely complementary to the portion of any one sequence from sequence numbers 125 to 207 that starts at the second nucleotide and ends at the eighth nucleotide, and a sequence that is incompletely complementary to the sequence that starts at the ninth nucleotide. In one preferred embodiment of the present invention, the target sequence is capable of binding to a short single-stranded RNA having any one sequence of SEQ ID NOs: 125 to 134. For example, the target sequence is a sequence that is fully complementary to any one sequence of SEQ ID NOs: 125 to 134, or a sequence that is fully complementary to a portion of any one sequence of SEQ ID NOs: 125 to 134 that starts at the second nucleotide and ends at the eighth nucleotide, and a sequence that is incompletely complementary to a sequence that starts at the ninth nucleotide. The short single-stranded RNA may have any one sequence of SEQ ID NOs: 125 to 207. In one preferred embodiment of the present invention, the short single-stranded RNA may have any one sequence of SEQ ID NOs: 125 to 134. The target sequence is a sequence that does not interfere with the translation of the target protein by the RNA molecule when the short single-stranded RNA is not bound to it, and may have the property that the translation of the target protein by the RNA molecule is inhibited when the short single-stranded RNA binds to the target sequence. RNA molecules may function as an OFF switch for protein translation, as the binding of short single-stranded RNA to a target sequence inhibits the translation of the target protein.

[0150] If the short single-stranded RNA is an RNA molecule with an artificial sequence, the target sequence has a sequence to which the artificial short single-stranded RNA can bind. When a composition for administering RNA molecules contains short single-stranded RNA that can bind to the target sequence, the RNA molecule after administration will have the short single-stranded RNA bound to the target sequence, resulting in a state where protein translation is inhibited in vivo. If the short single-stranded RNA is an RNA molecule with an artificial sequence, it may also be capable of binding complementaryly to the 2nd to 8th nucleotides from the 3' end of the target sequence contained in the target domain. If the short single-stranded RNA is an RNA molecule with an artificial sequence, the target sequence contained in the short single-stranded RNA target domain may be a sequence other than the short single-stranded RNA, for example, a sequence that does not bind to natural RNA molecules, specifically natural short single-stranded RNA such as miRNA.

[0151] It is acceptable for miRNAs, which are ubiquitously expressed in the tissue to which the RNA molecule was delivered, to bind to the target sequence of the administered RNA molecule, thereby inhibiting protein translation in vivo. miR21 can be used as a ubiquitously expressed miRNA, along with any of the following: hsa-miR-21-5p (miR21 in the specified examples, SEQ ID NO: 125), hsa-let-7a-5p (let7a in the specified examples, SEQ ID NO: 126), hsa-let-7b-5p (let7b in the specified examples, SEQ ID NO: 127), hsa-let-7c-5p (let7c in the specified examples, SEQ ID NO: 128), hsa-let-7d-5p (let7d in the specified examples, SEQ ID NO: 129), hsa-let-7e-5p (let7e in the specified examples, SEQ ID NO: 130), hsa-let-7f-5p (let7f in the specified examples, SEQ ID NO: 131), hsa-let-7g-5p (let7g in the specified examples, SEQ ID NO: 132), hsa-let-7i-5p (let7i in the specified examples, SEQ ID NO: 133), hsa-miR-23a-3p (miR23a in the examples herein, SEQ ID NO: 134).

[0152] The target sequence of the RNA molecule after administration is a sequence that binds to miR21, as shown in SEQ ID NO: 125, and may be, for example, the sequence shown in SEQ ID NO: 208, or a sequence in which the nucleotides in SEQ ID NO: 208 are appropriately replaced with modified nucleotides.

[0153] The target sequence of the RNA molecule after administration is a sequence that binds to let7a, as shown in SEQ ID NO: 126, and may be, for example, the sequence shown in SEQ ID NO: 210, or a sequence in which the nucleotides in SEQ ID NO: 210 are appropriately replaced with modified nucleotides.

[0154] The target sequence of the RNA molecule after administration is a sequence that binds to let7b, as shown in SEQ ID NO: 127, and may be, for example, the sequence shown in SEQ ID NO: 215, or a sequence in which the nucleotides in SEQ ID NO: 215 are appropriately replaced with modified nucleotides.

[0155] The target sequence of the RNA molecule after administration is a sequence that binds to let7c, as shown in SEQ ID NO: 128, and may be, for example, the sequence shown in SEQ ID NO: 216, or a sequence in which the nucleotides in SEQ ID NO: 216 are appropriately replaced with modified nucleotides.

[0156] The target sequence of the RNA molecule after administration is a sequence that binds to let7d, as shown in SEQ ID NO: 129, and may be, for example, the sequence shown in SEQ ID NO: 217, or a sequence in which the nucleotides in SEQ ID NO: 217 are appropriately replaced with modified nucleotides.

[0157] The target sequence of the RNA molecule after administration is a sequence that binds to let7e, as shown in SEQ ID NO: 130, and may be, for example, the sequence shown in SEQ ID NO: 218, or a sequence in which the nucleotides in SEQ ID NO: 218 are appropriately replaced with modified nucleotides.

[0158] The target sequence of the RNA molecule after administration is a sequence that binds to let7f as shown in SEQ ID NO: 131, and may be, for example, the sequence shown in SEQ ID NO: 219, or a sequence in which the nucleotides in SEQ ID NO: 219 are appropriately replaced with modified nucleotides.

[0159] The target sequence of the RNA molecule after administration is a sequence that binds to let7g as shown in SEQ ID NO: 132, and may be, for example, the sequence shown in SEQ ID NO: 220, or a sequence in which the nucleotides in SEQ ID NO: 220 are appropriately replaced with modified nucleotides.

[0160] The target sequence of the RNA molecule after administration is a sequence that binds to let7i, as shown in SEQ ID NO: 133, and may be, for example, the sequence shown in SEQ ID NO: 221, or a sequence in which the nucleotides in SEQ ID NO: 221 are appropriately replaced with modified nucleotides.

[0161] The target sequence of the RNA molecule after administration is a sequence that binds to miR23a, as shown in SEQ ID NO: 134, and may be, for example, the sequence shown in SEQ ID NO: 222, or a sequence in which the nucleotides in SEQ ID NO: 222 are appropriately replaced with modified nucleotides.

[0162] The target sequence of the RNA molecule after administration is a sequence that binds to miR122, as shown in SEQ ID NO: 141, and may be, for example, the sequence shown in SEQ ID NO: 272, or a sequence in which nucleotides in SEQ ID NO: 272 are appropriately replaced with modified nucleotides.

[0163] The target sequence of the RNA molecule after administration is a sequence that binds to miR126, as shown in SEQ ID NO: 142, and may be, for example, the sequence shown in SEQ ID NO: 273, or a sequence in which nucleotides in SEQ ID NO: 273 are appropriately replaced with modified nucleotides.

[0164] The target sequence of the RNA molecule after administration is a sequence that binds to miR142, as shown in SEQ ID NO: 144, and may be, for example, the sequence shown in SEQ ID NO: 274, or a sequence in which nucleotides in SEQ ID NO: 274 are appropriately replaced with modified nucleotides.

[0165] Short single-stranded RNA may be a ubiquitously expressed miRNA. In this specification, ubiquitously expressed means that its expression is uniform in each tissue within the target organism. A ubiquitously expressed miRNA can refer to a miRNA expressed in 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, 95% or more, 98% or more, or 99% or more of the tissues or cells within the target organism. Those skilled in the art can appropriately select a miRNA that is expressed in the tissue to which the RNA molecule is delivered from, for example, the miRNAs described in Rie et al. (Nature Biotechnology volume 35, pages 872-878 (2017)).

[0166] It is acceptable for a miRNA, which is expressed to a certain extent in the tissue to which the RNA molecule was delivered, to bind to the target sequence of the administered RNA molecule, thereby inhibiting protein translation in vivo. Any one of the following can be used as the miRNA that is expressed to a certain extent in the tissue to which the RNA molecule was delivered:

[0167] hsa-miR-21-5p (miR21 in the specified examples, SEQ ID NO: 125), hsa-let-7a-5p (let7a in the specified examples, SEQ ID NO: 126), hsa-let-7b-5p (let7b in the specified examples, SEQ ID NO: 127), hsa-let-7c-5p (let7c in the specified examples, SEQ ID NO: 128), hsa-let-7d-5p (let7d in the specified examples, SEQ ID NO: 129), hsa-let-7e-5p (let7e in the specified examples, SEQ ID NO: 130), hsa-let-7f-5p (let7f in the specified examples, SEQ ID NO: 131), hsa-let-7g-5p (let7g in the specified examples, SEQ ID NO: 132), hsa-let-7i-5p (let7i in the specified examples, SEQ ID NO: 133), hsa-miR-23a-3p (miR23a in the specified examples, SEQ ID NO: 134), hsa-miR-100-3p (SEQ ID NO: 135), hsa-miR-100-5p (SEQ ID NO: 136), hsa-miR-103a-1-5p (SEQ ID NO: 137), hsa-miR-103a-2-5p (SEQ ID NO: 138), hsa-miR-103a-3p (SEQ ID NO: 139), hsa-miR-122-3p (SEQ ID NO: 140), hsa-miR-122-5p (SEQ ID NO: 141), hsa-miR-126-3p (SEQ ID NO: 142), hsa-miR-126-5p (SEQ ID NO: 143), hsa-miR-142-3p (SEQ ID NO: 144), hsa-miR-142-5p (SEQ ID NO: 145), hsa-miR-143-3p (SEQ ID NO: 146), hsa-miR-143-5p (SEQ ID NO: 147), hsa-miR-15a-3p (SEQ ID NO: 148), hsa-miR-15a-5p (SEQ ID NO: 149), hsa-miR-15b-3p (SEQ ID NO: 150), hsa-miR-15b-5p (SEQ ID NO: 151), hsa-miR-16-1-3p (SEQ ID NO: 152), hsa-miR-16-2-3p (SEQ ID NO: 153), hsa-miR-16-5p (SEQ ID NO: 154),hsa-miR-181a-2-3p (SEQ ID NO: 155), hsa-miR-181a-3p (SEQ ID NO: 156), hsa-miR-181a-5p (SEQ ID NO: 157), hsa-miR-181b-2-3p (SEQ ID NO: 158), hsa-miR-181b-3p (SEQ ID NO: 159), hsa-miR-181b-5p (SEQ ID NO: 160), hsa-miR-191-3p (SEQ ID NO: 161), hsa-miR-191-5p (SEQ ID NO: 162), hsa-miR-195-3p (SEQ ID NO: 163), hsa-miR-195-5p (SEQ ID NO: 164), hsa-miR-202-3p (SEQ ID NO: 165), hsa-miR-202-5p (SEQ ID NO: 166), hsa-miR-205-3p (SEQ ID NO: 167), hsa-miR-205-5p (SEQ ID NO: 168), hsa-miR-23a-5p (SEQ ID NO: 169), hsa-miR-23b-3p (SEQ ID NO: 170), hsa-miR-23b-5p (SEQ ID NO: 171), hsa-miR-24-1-5p (SEQ ID NO: 172), hsa-miR-24-2-5p (SEQ ID NO: 173), hsa-miR-24-3p (SEQ ID NO: 174), hsa-miR-26a-1-3p (SEQ ID NO: 175), hsa-miR-26a-2-3p (SEQ ID NO: 176), hsa-miR-26a-5p (SEQ ID NO: 177), hsa-miR-26b-3p (SEQ ID NO: 178), hsa-miR-26b-5p (SEQ ID NO: 179), hsa-miR-27b-3p (SEQ ID NO: 180), hsa-miR-27b-5p (SEQ ID NO: 181), hsa-miR-28-3p (SEQ ID NO: 182), hsa-miR-28-5p (SEQ ID NO: 183), hsa-miR-29a-3p (SEQ ID NO: 184), hsa-miR-29a-5p (SEQ ID NO: 185), hsa-miR-320a-3p (SEQ ID NO: 186), hsa-miR-320a-5p (SEQ ID NO: 187), hsa-miR-423-3p (SEQ ID NO: 188), hsa-miR-423-5p (SEQ ID NO: 189),hsa-miR-484 (SEQ ID NO: 190), hsa-miR-497-3p (SEQ ID NO: 191), hsa-miR-497-5p (SEQ ID NO: 192), hsa-miR-500a-3p (SEQ ID NO: 193), hsa-miR-500a-5p (SEQ ID NO: 194), hsa-miR-532-3p (SEQ ID NO: 195), hsa-miR-532-5p (SEQ ID NO: 196), hsa-miR-769-3p (SEQ ID NO: 197), hsa-miR-769-5p (SEQ ID NO: 198), hsa-miR-92a-1-5p (SEQ ID NO: 199), hsa-miR-92a-2-5p (SEQ ID NO: 200), hsa-miR-92a-3p (SEQ ID NO: 201), hsa-miR-92b-3p (SEQ ID NO: 202), hsa-miR-92b-5p (SEQ ID NO: 203), hsa-miR-98-3p (SEQ ID NO: 204), hsa-miR-98-5p (SEQ ID NO: 205), hsa-miR-99b-3p (SEQ ID NO: 206), hsa-miR-99b-5p (SEQ ID NO: 207).

[0168] The RNA molecule contains an aptamer domain that can bind to a protein different from the target protein (target protein), and the protein that can bind to the aptamer domain (target protein) may be a protein that is present in the target organism.

[0169] The protein that can bind to the aptamer domain (target protein) may be a protein present in cells within the target organism. Examples of proteins that can bind to the aptamer domain (target protein) include enzymes, hormones, receptors, and ligands. The protein that can bind to the aptamer domain (target protein) may also be a protein present in the cytoplasm. The protein that can bind to the aptamer domain (target protein) may also be the intracellular domain of a protein present on the cell membrane. Those skilled in the art can select a protein expressed in the tissue to be delivered from an existing protein expression database (for example, https: / / www.proteinatlas.org / search / subcell_location:Aggresome,Cytosol,Cytoplasmic%20bodies,Rods%20&%20Rings) and use it as the target protein. Furthermore, by selecting a protein expressed in cells in a specific state in the tissue to be delivered, the translation of the RNA molecule of the present invention can be prevented from being inhibited in cells in that specific state. Examples of proteins expressed in cells under specific conditions include p16 and p21, which are expressed in senescent cells, and viral proteins (HBx and HBc) that are expressed only in hepatocytes infected with hepatitis B, but this is not an exhaustive list.

[0170] Examples of proteins present within cells are shown in Table 1, but are not limited to these. When the proteins in Table 1 are cytoplasmic proteins, the binding site of the aptamer domain to the protein is not particularly limited. When the proteins in Table 1 are cell membrane proteins, an aptamer domain capable of binding to the intracellular domain of the protein is used.

[0171]

[0172]

[0173] The binding of the aptamer domain to the protein may inhibit the binding of short single-stranded nucleic acids to the target sequence, thereby enabling translation of the target protein by the sequence encoding the target protein. The binding of the aptamer domain to the protein may inhibit the binding of short single-stranded nucleic acids to the target sequence that is not bound to them, thereby maintaining translation of the target protein by the sequence encoding the target protein. The binding of the aptamer domain to the protein may promote the dissociation of short single-stranded nucleic acids bound to the target sequence, thereby enabling translation of the target protein by the sequence encoding the target protein. The RNA molecule has a configuration in which the binding of the aptamer domain to the protein does not affect the translation of the target protein by the sequence encoding the target protein. The configuration of the aptamer domain that does not affect the translation of the target protein by the sequence encoding the target protein refers to a configuration of the aptamer domain in which the expression level of the target protein is equivalent when an RNA molecule containing the sequence encoding the target protein and an aptamer domain is introduced into the same type of cell as an RNA molecule containing the sequence encoding the target protein and an aptamer domain. The aptamer domain of the RNA molecule may be contained in the 3'UTR.

[0174] The binding of short single-stranded nucleic acids to target sequences can be carried out using biological or chemical measurement techniques known in the art. For example, assay systems using reporter genes, or methods combining the isolation of physical binding complexes with nucleic acid quantification can be used.

[0175] One quantitative measurement method using a reporter gene (reporter assay) involves using a recombinant vector in which a target sequence of a short single-stranded nucleic acid is ligated to the 3' untranslated region (3'UTR) of a gene encoding a detectable signal-emitting protein such as firefly luciferase, sea urchin luciferase, or green fluorescent protein (GFP). In this method, the vector and the target short single-stranded nucleic acid are co-introduced into cells, and the luminescence intensity or fluorescence intensity derived from the reporter protein is measured. When the short single-stranded nucleic acid binds to the target sequence and translational repression or mRNA degradation occurs, a decrease in signal intensity is observed. Therefore, the strength of binding and the repressive effect can be quantitatively evaluated using the relative signal reduction rate compared to the control group as an indicator.

[0176] Furthermore, as a method for directly quantifying the amount of physical binding, a combination of RNA immunoprecipitation (RIP) or pull-down method and quantitative reverse transcription PCR (RT-qPCR) or digital PCR may be used. Specifically, the short single-stranded nucleic acid-mRNA complex is specifically recovered using an antibody against the Argonaut (AGO) protein that forms a complex with short single-stranded nucleic acids, or by using short single-stranded nucleic acids labeled with biotin, etc. Subsequently, RNA is extracted from the recovered complex, and by performing real-time PCR using a primer for the target mRNA, it is possible to absolutely or relatively quantify the number of mRNA molecules that were bound to the short single-stranded nucleic acid.

[0177] Furthermore, if a more precise quantification of intermolecular interactions is required, physicochemical or optical measurement techniques such as surface plasmon resonance (SPR), isothermal titration calorimetry (ITC), or fluorescence resonance energy transfer (FRET) may be used to calculate the bond dissociation constant (Kd value) and bond kinetic parameters.

[0178] The method for measuring whether the binding of the aptamer domain to the target protein inhibits the binding of short single-stranded nucleic acids to the target sequence is not particularly limited. For example, such measurements can be performed by functional assays using cells (evaluation of recovery at the translational level), biochemical assays using purified molecules (evaluation of physical competition), or a combination thereof.

[0179] For functional evaluation using cells, a competitive reporter assay is preferably used. Specifically, an expression vector is constructed by ligating a sequence containing an aptamer domain and a short single-stranded nucleic acid target sequence to the untranslated region (UTR) of a reporter gene (e.g., luciferase or GFP). When this vector is introduced into cells expressing the target short single-stranded nucleic acid, the expression of the reporter gene is usually suppressed. When a target protein capable of binding to the aptamer domain is added to this system (e.g., co-introduction of the protein expression vector or addition of the protein), the reporter signal (luminescence or fluorescence) is measured to see if it is restored or increased. If the signal is restored in a concentration-dependent manner with respect to the target protein, it can be determined that the binding or function of the short single-stranded nucleic acid was inhibited by the binding of the protein.

[0180] Furthermore, gel shift assays (EMSA) or surface plasmon resonance (SPR) analysis may be used as methods to directly evaluate intermolecular physical competition or steric hindrance. When using EMSA, an RNA fragment containing the aptamer domain and short single-stranded nucleic acid target sequence, a short single-stranded nucleic acid (or a complex with AGO protein), and the target protein are mixed, incubated, and then electrophoresis is performed. By confirming that the band derived from the short single-stranded nucleic acid-RNA complex disappears or decreases upon addition of the target protein, and that a band derived from the protein-RNA complex appears instead, the substitution of binding can be visualized. When using SPR, the inhibition rate of short single-stranded nucleic acid binding by target protein binding can be quantitatively calculated by comparing the binding response when short single-stranded nucleic acid is passed over RNA containing the aptamer domain and short single-stranded nucleic acid target sequence immobilized on a sensor chip with the binding response when short single-stranded nucleic acid is passed over after the target protein has been pre-bound.

[0181] Furthermore, RNA immunoprecipitation (RIP) may be applied to confirm intracellular competition. For example, immunoprecipitation can be performed on cells overexpressing the target protein and control cells using an antibody against a short single-stranded nucleic acid-binding protein (such as AGO protein), and the co-precipitated RNA can be recovered. If the amount of target mRNA bound to the AGO protein is reduced compared to the control in the presence of the target protein, this data can be used to suggest that the target protein inhibits access to the short single-stranded nucleic acid complex within the cell.

[0182] The binding of the aptamer domain to the target protein inhibits the binding of short single-stranded nucleic acids to the target sequence, which means that the binding of short single-stranded nucleic acids to the target sequence in the presence of the target protein is 90% or less, 80% or less, 70% or less, 60% or less, 50% or less, 40% or less, 30% or less, 20% or less, 10% or less, 5% or less, 4% or less, 3% or less, 2% or less, or 1% or less compared to the binding of short single-stranded nucleic acids to the target sequence in the absence of the target protein.

[0183] The protein binding of the aptamer domain may be specific. This specificity means that the aptamer has high affinity and selectivity for the target protein. The aptamer constituting the aptamer domain may be an aptamer that exhibits affinity for the target protein with a dissociation constant (Kd) of 1000 nM or less, 800 nM or less, 500 nM or less, 300 nM or less, 200 nM or less, 100 nM or less, 80 nM or less, 60 nM or less, 50 nM or less, 30 nM or less, 20 nM or less, 10 nM or less, 8 nM or less, 5 nM or less, 3 nM or less, 2 nM or less, or 1 nM or less. The aptamer constituting the aptamer domain preferably exhibits affinity for the target protein with a dissociation constant (Kd) of 50 nM or less, most preferably 5 nM or less. The aptamers constituting the aptamer domain exhibit low affinity for proteins other than the target protein, and the difference in affinity between the two may be 5 times or more, 10 times or more, 50 times or more, 100 times or more, 200 times or more, 300 times or more, 500 times or more, 800 times or more, or 1000 times or more. It is most preferable that the affinity of the aptamer for the target protein is 50 times or more than the affinity for proteins other than the target protein. It is preferable that the affinity for structurally similar proteins or other proteins belonging to the same family is at least 100 times lower compared to the target protein.

[0184] This high degree of specificity is achieved and validated by multiple methods. First, multi-round SELEX assays are used to eliminate aptamer domains exhibiting nonspecific binding, rigorously selecting only sequences that specifically bind to the target protein. Next, competitive binding assays using radioisotopes or fluorescent labeling are used to comparatively quantify the binding affinity between the target protein and structurally similar proteins. Furthermore, surface plasmon resonance (SPR) systems such as Biacore are used to measure the interaction between the aptamer and various proteins in real time, thereby evaluating binding specificity.

[0185] In addition, we confirmed using a pull-down assay that aptamers can specifically capture only the target protein from complex protein mixtures (e.g., cell lysates), demonstrating that specific detection of target proteins is possible on cells and tissue sections using fluorescently labeled aptamers.

[0186] The aptamers comprising the aptamer domain described herein can be prepared by known methods. In this regard, for example, U.S. Patent Application No. 07 / 536,428, U.S. Patent No. 5,475,096 and U.S. Patent No. 5,270,163 (see also WO91 / 198 13) are incorporated herein by reference.

[0187] One known method for obtaining aptamers is the SELEX method. The SELEX method utilizes a large library or pool of single-stranded oligonucleotides containing randomized sequences as a starting point, where the oligonucleotides can be modified or unmodified, DNA, RNA, or DNA / RNA hybrids. In some examples, the pool contains 100% random or partially random oligonucleotides.

[0188] The oligonucleotide pool used in the SELEX method preferably contains randomized sequence portions and fixed sequences necessary for efficient amplification. Generally, the starting pool oligonucleotides contain fixed 5' and 3' terminal sequences located on the sides of the internal regions of 30 to 50 random nucleotides. Randomized nucleotides can be generated by several methods, including chemosynthesis and size selection from randomly cleaved cellular nucleic acids. Sequence mutations in the test nucleic acid can also be introduced or amplified by mutagenesis before or during selection / amplification repeats.

[0189] The random sequence portion of the oligonucleotide can be of any length and may contain ribonucleotides and / or deoxyribonucleotides, and may contain modified or unnatural nucleotides or nucleotide analogs. In this regard, U.S. Patents 5,958,691; 5,660,985; 5,958,691; 5,698,687; 5,817,635; 5,672,695 and PCT Publication WO92 / 07065 are incorporated herein by reference. Furthermore, the randomized sequences used in the SELEX method can be prepared by general synthesis carried out on an automated DNA synthesizer, for example, 10 14 ~10 16 This allows for the generation of individual molecules. A sufficiently large region of random sequences during sequence design increases the likelihood that each synthesized molecule will represent a unique sequence.

[0190] In the SELEX method, when an RNA library is used as the starting library, it is generally produced and purified by in vitro transcription of a DNA library using T7 RNA polymerase or modified T7 RNA polymerase. The library is then mixed with the target under binding-favorable conditions and subjected to repeated binding, splitting, and amplification steps using the same general selection scheme to achieve any desired criteria of binding affinity and selectivity. More specifically, starting from a mixture containing a starting pool of nucleic acids, the SELEX method includes (a) contacting the mixture with the target under binding-favorable conditions; (b) splitting unbound nucleic acids from nucleic acids specifically bound to the target molecule; (c) separating the nucleic acid-target complex; (d) amplifying the nucleic acids separated from the nucleic acid-target complex to obtain a ligand-amplified nucleic acid mixture; and (e) repeating the binding, splitting, separation, and amplification steps for a desired number of cycles to obtain a nucleic acid ligand with high specificity and high affinity for the target molecule. If an RNA aptamer is selected, the SELEX method further includes (i) a step of reverse transcribing the nucleic acid separated from the nucleic acid-target complex before amplification in step (d); and (ii) a step of transcribing the amplified nucleic acid from step (d) before restarting the process.

[0191] Improved versions of the SELEX method are also known. In this regard, U.S. Patents 5,707,796, 5,763,177, 5,567,588, 5,861,254, 5,705,337, and 5,580,737 are incorporated herein by reference.

[0192] Various engineering techniques can be used to improve the binding activity and specificity of aptamers. For example, sequence optimization using the SELEX method can select sequences with high affinity for the target molecule, and binding activity can be improved by in vitro evolution. Furthermore, chemical modifications such as modification of the 2' position of nucleotides or introduction of phosphorothioate bonds can improve nuclease resistance and extend the blood half-life, thereby potentially improving binding activity.

[0193] Structural stabilization is also an important technique. By stabilizing secondary structures such as G-quadruplex structures and stem-loop structures, the three-dimensional structure of the aptamer can be maintained and its binding activity improved. For example, structural stabilization can be achieved by introducing cross-linking nucleotides such as LNA and UNA. Furthermore, by linking two aptamer domains that bind to different targets to create a bispecific aptamer, the specificity of binding can be improved.

[0194] Immobilizing aptamers onto polymer matrices with artificial binding pockets designed to match the shape of target molecules using molecular imprinting technology is also effective in improving binding specificity. In addition, by utilizing computer-aided design, molecular dynamics simulations, and machine learning algorithms to predict interactions with targets and designing optimal sequences and structures, binding activity and specificity can be improved.

[0195] By using these engineering techniques individually or in combination, it is possible to significantly improve the binding activity and specificity of aptamers, leading to the creation of more effective aptamers.

[0196] In the state where a protein is bound to the aptamer domain, steric obstruction occurs in the short single-stranded nucleic acid's access to the target sequence on the RNA molecule of the present invention. This reduces the amount of short single-stranded nucleic acid that can bind to the RNA molecule of the present invention, thereby reducing the amount of the RNA molecule of the present invention that is degraded, or making it less likely for the translation of the RNA molecule of the present invention into the target protein to be inhibited, and thus increasing the amount of translation of the target protein. As a result, the RNA molecule of the present invention has the function of switching on the translation of the encoded protein in the delivered cell.

[0197] When a protein is bound to the aptamer domain, it becomes difficult for the short single-stranded RNA to seed-match with the target sequence on the RNA molecule of the present invention. This reduces the amount of short single-stranded RNA that can bind to the RNA molecule of the present invention, thus reducing the amount of the RNA molecule of the present invention that is degraded, or suppressing the inhibition of translation from the RNA molecule of the present invention to the target protein, thereby increasing the amount of translation of the target protein. In this way, the RNA molecule of the present invention has the function of switching on the translation of the encoded protein in the delivered cell.

[0198] The target sequence contained in the RNA molecule may be adjacent to the aptamer sequence. In other words, in one embodiment of the RNA molecule of the present invention, the aptamer sequence and the target sequence may be physically adjacent to each other. Specifically, the aptamer sequence and the target sequence may be consecutively aligned in the primary structure of the RNA molecule and directly linked without the interposition of other functional sequences or structural elements between the two sequences. In other words, in one embodiment of the RNA molecule of the present invention, the aptamer sequence and the target sequence may be directly linked without overlap. Direct linkage of the aptamer sequence and the target sequence without overlap can be rephrased as the last nucleotide of the aptamer sequence being covalently bonded to the first nucleotide of the target sequence when viewed from the 5' to 3' direction or from the 3' to 5' direction of the same RNA strand. The aptamer sequence may be located on the 5' end side of the target sequence, or on the 3' end side of the target sequence. This relative positional relationship can be appropriately selected depending on the characteristics of the sequences used and the expected biological effects.

[0199] One or more nucleotides in an aptamer sequence contained within an RNA molecule may be included as part of a target sequence contained within the same RNA molecule. That is, one or more nucleotides in an aptamer sequence may constitute a part of a target sequence. One or more nucleotides in an aptamer sequence that constitute a part of a target sequence may be located at the 3' end, the 5' end, or at a non-terminal location. Furthermore, one or more nucleotides in an aptamer sequence that constitute a part of a target sequence may or may not be contiguous.

[0200] One or more nucleotides in a target sequence contained within an RNA molecule may be included as part of an aptamer sequence contained within the same RNA molecule. That is, one or more nucleotides in a target sequence may constitute part of an aptamer sequence. One or more nucleotides in a target sequence that constitute part of an aptamer sequence may be located at the 3' end, the 5' end, or at a non-terminal location of the target sequence. Furthermore, one or more nucleotides in a target sequence that constitute part of an aptamer sequence may be consecutive or not.

[0201] In this specification, one or more nucleotides that simultaneously constitute a portion of the aptamer sequence that makes up the aptamer domain and a portion of the target sequence contained in the short single-stranded nucleic acid target domain may be referred to as the "overlap portion of the aptamer sequence and target sequence".

[0202] One or more nucleotides at the 3' end of an aptamer sequence contained in an RNA molecule constitute a part of a target sequence contained in the same RNA molecule, the 5' end of the aptamer sequence has a sequence complementary to the one or more nucleotides, and the 3' and 5' ends of the aptamer sequence may be capable of forming a stem.

[0203] One or more nucleotides at the 3' end of the aptamer sequence may constitute the 5' end of the target sequence. One or more nucleotides at the 3' end of the aptamer sequence may constitute the 3' end of the target sequence. One or more nucleotides at the 3' end of the aptamer sequence may constitute a non-terminal portion of the target sequence.

[0204] When multiple nucleotides at the 3' end of an aptamer sequence constitute part of a target sequence, and the 5' end of the aptamer sequence has a sequence complementary to these multiple nucleotides, and a stem can be formed between the 3' and 5' ends of the aptamer sequence, it is preferable that the multiple nucleotides consist of 3 to 8 nucleotides.

[0205] One or more nucleotides at the 5' end of an aptamer sequence contained in an RNA molecule constitute a part of a target sequence contained in the same RNA molecule, the 3' end of the aptamer sequence has a sequence complementary to the one or more nucleotides, and the 3' and 5' ends of the aptamer sequence may be capable of forming a stem.

[0206] One or more nucleotides at the 5' end of the aptamer sequence may constitute the 3' end of the target sequence. One or more nucleotides at the 5' end of the aptamer sequence may constitute the 5' end of the target sequence. One or more nucleotides at the 5' end of the aptamer sequence may constitute a non-terminal portion of the target sequence.

[0207] When a plurality of nucleotides at the 5' end of an aptamer sequence constitute a part of the target sequence, and the 3' end of the aptamer sequence has a sequence complementary to the plurality of nucleotides, and a stem can be formed at the 3' and 5' ends of the aptamer sequence, it is preferable that the plurality of nucleotides consist of 3 to 8 nucleotides. More preferably, the plurality of nucleotides consist of 3 to 6 nucleotides.

[0208] The target sequence contained in an RNA molecule may also be contained in the aptamer sequence of the same RNA molecule. In other words, the entire target sequence in an RNA molecule is part of the aptamer sequence. Within the aptamer sequence, there are regions that have sequences complementary to a part of the target sequence, and these sequences can form a stem.

[0209] This disclosure provides a linear RNA molecule comprising: a sequence encoding a protein of interest; an aptamer domain capable of binding to a protein different from the protein of interest (a target protein); and a short single-stranded nucleic acid target domain consisting of a target sequence of a short single-stranded nucleic acid, wherein the binding of the aptamer domain to the target protein inhibits the binding of the target sequence to the short single-stranded nucleic acid; the sequences located at the 5' end and 3' end of the aptamer domain are capable of forming a stem; the sequences located at the 5' end and 3' end of the aptamer domain, capable of forming a stem, each independently have 3 to 10 nucleotides; and one or more nucleotides located at the 5' end of the aptamer domain constitute a part of the target sequence that binds to the short single-stranded nucleic acid.

[0210] This disclosure provides a cyclic RNA molecule comprising a sequence encoding a protein of interest, an aptamer domain capable of binding to a protein different from the protein of interest (a target protein), and a short single-stranded nucleic acid target domain consisting of the target sequence, wherein the binding of the aptamer domain to the target protein inhibits the binding of the target sequence to the short single-stranded nucleic acid, the sequence located at the 5' end and the sequence located at the 3' end of the aptamer domain are capable of forming a stem, the sequence located at the 5' end and the sequence located at the 3' end of the aptamer domain each independently have 3 to 10 nucleotides, and one or more nucleotides located at the 5' end of the aptamer domain constitute a part of the short single-stranded nucleic acid target domain.

[0211] A composition containing RNA molecules may have RNA molecules forming complexes or aggregates. The composition containing RNA molecules may further contain carriers.

[0212] The composition containing the RNA molecule may be a composition that does not contain nucleic acids other than the RNA molecule of the present invention. In addition to the RNA molecule of the present invention, the composition containing the RNA molecule may further contain a precursor of a short single-stranded nucleic acid that can bind to the target sequence of the RNA molecule of the present invention. The precursor of the short single-stranded nucleic acid is a short single-stranded RNA precursor or a short single-stranded DNA precursor. The short single-stranded RNA precursor is preferably shRNA or siRNA.

[0213] RNA molecules can be complexed or associated with one or more (poly)cationic compounds, preferably (poly)cationic polymers, (poly)cationic peptides, or proteins, such as protamine, (poly)cationic polysaccharides, and / or (poly)cationic lipids. RNA molecules can be complexed or associated with lipids (particularly cationic and / or neutral lipids) to form one or more liposomes, lipoplexes, lipid nanoparticles, or nanoliposomes.

[0214] In lipid nanoparticles containing RNA molecules, the lipid nanoparticles (LNPs) may include (a) RNA molecules, (b) cationic lipids, (c) an aggregation inhibitor (such as polyethylene glycol (PEG) lipids or PEG-modified lipids), (d) non-cationic lipids (such as neutral lipids), and / or (e) optionally sterols.

[0215] The LNP comprises any cationic lipid suitable for forming lipid nanoparticles, the cationic lipid may be positively charged to a level at which the lipid nanoparticles have a physiological pH. The cationic lipid may be an aminolipid. In this specification, the term "aminolipid" means a lipid having one or two fatty acids or fatty alkyl chains and an amino group (including alkylamino or dialkylamino groups) that can be protonated to form a cationic lipid at a physiological pH.

[0216] Cationic lipids include N,N-dioleyl-N,N-dimethylammonium chloride (DODAC), N,N-distearyl-N,N-dimethylammonium bromide (DDAB), 1,2-dioleoyltrimethylammoniumpropane chloride (DOTAP) (also known as N-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride and 1,2-dioleyloxy-3-trimethylaminopropane chloride salt), and N-(1-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride. (DOTMA), N,N-dimethyl-2,3-dioleyloxy)propylamine (DODMA), 1,2-dilinoleyloxy-N,N-dimethylaminopropane (DLinDMA), 1,2-dilinolelenyloxy-N,N-dimethylaminopropane (DLenDMA), 1,2-di-γ-linolelenyloxy-N,N-dimethylaminopropane (γ-DLenDMA), 1,2-dilinoleylcarbamoyloxy-3-dimethylaminopropane (DLin-C-DAP), 1,2-dilinoleyloxy-3-(dimethylamino)acetoxypropane (DL in-DAC), 1,2-dilinoleyloxy-3-morpholinopropane (DLin-MA), 1,2-dilinoleyl-3-dimethylaminopropane (DLinDAP), 1,2-dilinoleylthio-3-dimethylaminopropane (DLin-S-DMA), 1-linoleyl-2-linoleyloxy-3-dimethylaminopropane (DLin-2-DMAP), 1,2-dilinoleyloxy-3-trimethylaminopropane chloride (DLin-TMA.Cl), 1,2-dilinoleyl-3-trimethylaminopropane chloride (DLin-TAP. Cl), 1,2-dilinoleyloxy-3-(N-methylpiperazino)propane (DLin-MPZ), or 3-(N,N-dilinoleylamino)-1,2-propanediol (DLinAP), 3-(N,N-dioleylamino)-1,2-propanediol (DOAP), 1,2-dilinoleyloxo-3-(2-N,N-dimethylamino)ethoxypropane (DLin-EG-DMA), 2,2-dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA) or its analog, (3aR,5s,6aS)-N,N-dimethyl-2,2-di((9Z,12Z)-octadeca-9,12-dienyl)tetrahydro-3aH-cyclopenta[d][1,3]dioxol-5-amine, (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl-4-dimethylamino)butanoate (MC3), 1,1'-(2-(4-(2-((2-(bis(2-hydroxydodecyl)amino)ethyl)(2-hydroxydodecyl)amino)ethyl)piperazine-1-yl)ethylazanejyl)didodecane-2-ol (C12-200), 2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLin-K-C2-DMA) Examples include 2,2-dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA), (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl-4-(dimethylamino)butanoate (DLin-M-C3-DMA), 3-((6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,3-1-tetraen-19-yloxy)-N,N-dimethylpropane-1-amine (MC3 ether), and 4-((6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yloxy)-N,N-dimethylbutane-1-amine (MC4 ether).

[0217] Examples of cationic lipids include N,N-distearyl-N,N-dimethylammonium bromide (DDAB), 3P-(N-(N',N'-dimethylaminoethane)-carbamoyl)cholesterol (DC-Chol), N-(1-(2,3-dioleyloxy)propyl)-N-2-(sperminecarboxamide)ethyl)-N,N-dimethylammonium trifluoroacetate (DOSPA), dioctadecylamideglycylcarboxyspermine (DOGS), 1,2-dioleoyl-sn-3-phosphoethanolamine (DOPE), 1,2-dioleoyl-3-dimethylammoniumpropane (DODAP), N-(1,2-dimyristyloxyprop-3-yl)-N,N-dimethyl-N-hydroxyethylammonium bromide (DMRIE), and 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (XTC). Furthermore, cationic lipids such as lipofectin (including DOTMA and DOPE available from GIBCO / BRL) and lipofectamine (including DOSPA and DOPE available from GIBCO / BRL) are cited as examples.

[0218] LNP may contain two or more cationic lipids. The selection of cationic lipids can be appropriately made by those skilled in the art to which the present invention belongs, and may be determined based on properties such as amine pKa, chemical stability, half-life in circulation, half-life in tissue, net accumulation in tissue, or toxicity.

[0219] Cationic lipids may be present in LNPs in proportions of approximately 20 mol% to approximately 70 or 75 mol%, or approximately 45 to approximately 65 mol%, or approximately 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, or approximately 70 mol% of the total lipids present. LNPs may contain approximately 25% to approximately 75% cationic lipids on a molar basis, for example, approximately 20% to approximately 70%, approximately 35% to approximately 65%, approximately 45% to approximately 65%, approximately 60%, approximately 50%, or approximately 40% cationic lipids on a molar basis (when the total number of moles of lipids in lipid nanoparticles is considered 100%). The ratio of cationic lipids to nucleic acids may be approximately 3 to approximately 15, for example, approximately 5 to approximately 13 or approximately 7 to approximately 11.

[0220] Liposomes may have an N:P ratio (N:P ratio) of nitrogen atoms in the cationic lipid to phosphate in the RNA of 1:1 to 20:1. Liposomes may have an N:P ratio greater than 20:1 or less than 1:1.

[0221] LNP may contain noncationic lipids. In this specification, the term "noncationic lipid" means neutral lipid, anionic lipid, or amphiphilic lipid. Examples of neutral lipids include diacylphosphatidylcholine, diacylphosphatidylethanolamine, ceramide, sphingomyelin, dihydrosphingomyelin, kephalin, and cerebroside.

[0222] The neutral lipid may contain saturated fatty acids having a carbon chain length of C10 to C20. Alternatively, neutral lipids containing monounsaturated fatty acids having a carbon chain length of C10 to C20 may be used. Furthermore, neutral lipids having a mixture of saturated and unsaturated fatty acid chains may be used.

[0223] Furthermore, as neutral lipids, distearoyl phosphatidylcholine (DSPC), dioleoyl phosphatidylcholine (DOPC), dipalmitoyl phosphatidylcholine (DPPC), dioleoyl phosphatidylglycerol (DOPG), dipalmitoyl phosphatidylglycerol (DPPG), dioleoyl-phosphatidylethanolamine (DOPE), palmitoyloleoyl phosphatidylcholine (POPC), palmitoyloleoyl phosphatidylethanolamine (POPE), dioleoyl-phosphatidylethanolamine Examples include mine-4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), dimyristoylphosphatidylcholine (DMPC), distearoylphosphatidylethanolamine (DSPE), SM, 16-O-monomethylPE, 16-O-dimethylPE, 18-1-transPE, 1-stearoyl-2-oleoylphosphatidiethanolamine (SOPE), and cholesterol.

[0224] LNP may contain amphiphilic lipids. In this specification, "amphiphilic lipid" means any suitable material in which the hydrophobic portion of the lipid material is oriented towards the hydrophobic phase and the hydrophilic portion is oriented towards the aqueous phase. Amphiphilic lipids generally include, but are not limited to, phospholipids, aminolipids, and sphingolipids. Representative phospholipids include sphingomyelin, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidic acid, palmitoyloleoylphosphatidylcholine, lysophosphatidylcholine, lysophosphatidylethanolamine, dipalmitoylphosphatidylcholine, dioleoylphosphatidylcholine, distearoylphosphatidylcholine, or dilinoleoylphosphatidylcholine. Furthermore, sphingolipids, the sphingoglycolipid family, diacylglycerols, and beta-acyloxy acids are also exemplified as amphiphilic lipids.

[0225] Noncationic lipids may be present in LNPs in proportions of approximately 5 mol% to approximately 90 mol%, approximately 5 mol% to approximately 10 mol%, approximately 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, or approximately 90 mol% of the total lipids present.

[0226] LNPs contain approximately 0% to 15% or 45% of neutral lipids on a molar basis, for example, approximately 3% to 12% or approximately 5% to 10%. For example, LNPs may contain approximately 15%, 10%, 7.5%, or 7.1% of neutral lipids on a molar basis (assuming the total number of moles of lipids in the LNP is 100%).

[0227] LNP may contain sterols, preferably cholesterol. Sterols may be present in proportion to about 10 mol% to about 60 mol% or about 25 mol% to about 40 mol% of LNP. Sterols may be present in proportion to about 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, or about 60 mol% of the total lipids present in LNP. LNP may contain sterols in proportion to about 5% to about 50% on a molar basis (when the total number of moles of lipids in LNP is 100%), for example, about 15% to about 45%, about 20% to about 40%, about 48%, about 40%, about 38.5%, about 35%, about 34.4%, about 31.5%, or about 31% on a molar basis.

[0228] The composition containing RNA molecules may further contain an aggregation inhibitor, which is a lipid capable of inhibiting aggregation. Examples of lipids that can be used as aggregation inhibitors include polyethylene glycol (PEG)-modified lipids, monosialoganglioside G ml, and polyamide oligomers (PAOs) as described in U.S. Patent No. 6,320,017.

[0229] Examples of flocculation inhibitors include PEG-diacylglycerol (DAG), PEG-dialkylglycerol, PEG-dialkyloxypropyl (DAA), PEG-phospholipid, PEG-ceramide (Cer), or mixtures thereof (such as PEG-Cer14 or PEG-Cer20). Examples of PEG-DAA conjugates include PEG-dilauryloxypropyl (C12), PEG-dimyristyloxypropyl (C14), PEG-dipalmityloxypropyl (C16), or PEG-distearyloxypropyl (C18). Other PEGylated lipids include polyethylene glycol-didimyristoylglycerol (C14-PEG or PEG-C14 (PEG has an average molecular weight of 2000 Da) (PEG-DMG); (R)-2,3-bis(octadecyloxy)propyl-1-(methoxypoly(ethylene glycol)2000)propylcarbamate) (PEG-DSG); PEG-carbamoyl-1,2-dimyristyloxypropylamine (PEG has an average molecular weight of 2000 Da) The following are included: (PEG-cDMA) having a uniform molecular weight; N-acetylgalactosamine-((R)-2,3-bis(octadecyloxy)propyl-1-(methoxypoly(ethylene glycol)2000)propylcarbamate))(GalNAc-PEG-DSG); mPEG(mw2000)-diastearoylphosphatidyl-ethanolamine (PEG-DSPE); and polyethylene glycol-dipalmitoylglycerol (PEG-DPG). The flocculation inhibitor is preferably PEG-DMG. The flocculation inhibitor is also preferably PEG-cDMA.

[0230] DNA Molecules In this disclosure, DNA molecules encoding the RNA molecules described herein are also provided. When the RNA molecule of the present invention is mRNA, the DNA molecule encoding the RNA molecule has a DNA sequence capable of transcribing an RNA sequence that does not contain a cap structure and poly(A). The DNA molecule is transcribed into an RNA molecule within the delivered cell, and a cap structure and poly(A) are added. The DNA molecule may also be plasmid DNA. The DNA molecule encoding the RNA molecule described herein may be designed to generate the RNA molecule in vivo by transcription after being administered to a subject.

[0231] In lipid nanoparticles containing DNA molecules, the lipid nanoparticles (LNPs) may include (a) DNA molecules, (b) cationic lipids, (c) an aggregation inhibitor (such as polyethylene glycol (PEG) lipids or PEG-modified lipids), (d) non-cationic lipids (such as neutral lipids), and / or (e) optionally, sterols. The DNA molecules may be double-stranded linear DNA, single-stranded linear DNA, or plasmid DNA.

[0232] A DNA vector incorporates a DNA molecule. The DNA molecule may be a DNA vector designed to transcribe the target RNA sequence. The DNA vector may include a promoter, multiplexing sites (MCS), origin of replication (ORI), and terminator as components. The DNA vector may be a viral vector or a plasmid vector. Examples of viral vectors include adenoviruses, lentiviruses, and AAV (adeno-associated virus). A plasmid vector designed to transcribe the target RNA sequence may be contained within a carrier. Examples of carriers include lipid nanoparticles and liposomes.

[0233] Method for expressing a target protein / Method for controlling the expression of a target protein RNA molecules described in [A1] to [A42] above are produced from DNA molecules by transcription, and these RNA molecules may have the functions of both an OFF switch and an ON switch for protein translation as described above.

[0234] By using the RNA molecules of the present disclosure or DNA molecules that produce the RNA molecules of the present disclosure, even when a carrier such as an LNP delivers nucleic acids to unintended tissues, by controlling the translation level, the expression of the target protein in the unintended tissues can be suppressed, and the risk of side effects due to the expression of the target protein in the unintended tissues can be suppressed. The RNA molecules of the present disclosure serve as triggers for expressing proteins present in cells, and there is no need to administer triggers for expression control from the outside. "Autonomous therapy" is possible, which reads the state of the cells themselves and automatically switches the expression of therapeutic proteins. The RNA molecules of the present disclosure can tune the responsiveness of expression control by adjusting the positional relationship between the aptamer domain and the short single-stranded nucleic acid target domain (such as whether there are adjacent or overlapping nucleotides, the number of overlapping nucleotides, the position and length of the antisense with respect to the overlapping nucleotides, etc.). Therefore, they are molecules with high design flexibility. The RNA molecules of the present disclosure can control expression not only for linear mRNAs but also for circular RNAs, have versatility in being loaded onto various types of RNAs, and can simultaneously achieve stability and strict expression control.

[0235] [Example 1] Synthesis of mRNA Using a plasmid containing the DNA sequence serving as a template for each mRNA as a template, PCR (TOYOBO, KOD One (registered trademark) PCR Master Mix) was performed using the IVT_fw primer (SEQ ID NO: 1) and the IVT_rev primer (SEQ ID NO: 2). Subsequently, DpnI (New England Biolabs) was added to the reaction solution to remove the plasmid, and then the DNA of the PCR product was purified using the QIAquick (registered trademark) PCR Purification Kit (Qiagen).

[0236] Using the PCR product as a template, MEGAscript TMmRNA was synthesized using the T7 Transcription Kit (Invitrogen). During this process, CleanCap® Reagent AG (TriLink BioTechnologies) was added as a cap analog to a final concentration of 4 mM, and N1-methyl-pseudridine-5'-triphosphate (TriLink BioTechnologies) was added instead of the UTP included in the kit. Subsequently, rAPid Alkaline Phosphatase (Roche) and TURBO were used to degrade the DNA in the reaction mixture. TM DNase (Invitrogen) was added, and the reaction mixture was incubated at 37°C for 30 minutes. Subsequently, mRNA was purified using the RNeasy® mini kit (Qiagen). The mixture was then stored at -80°C.

[0237] [Example 2] Evaluation of L7Ae-dependent increase in translation of mRNA with an L7Ae binding sequence adjacent to the miR21 binding sequence. As shown in Figure 2, EGFP-encoded mRNA (Control EGFP, SEQ ID NO: 6), EGFP-encoded mRNA with a miR21 binding sequence (SEQ ID NO: 108) (EGFP_Tg21, SEQ ID NO: 7), and EGFP-encoded mRNA with an L7Ae binding sequence (hereinafter also called CDmini, SEQ ID NO: 112) [Citation: Saito H. et al., Nature Chemical Biology volume 6, pages 71-78 (2010)] on the 5' side of the miR21 binding sequence (EGFP_CDmini_Tg21, SEQ ID NO: 8) (various EGFP-encoded mRNAs can be hereinafter referred to as "EGFP mRNA") were created, and experiments were conducted using HeLa cells (ATCC, Cat.No. CCL-2) to investigate the changes in translation levels of these constructs in the presence of L7Ae.

[0238] First, HeLa cells were seeded in 12-well plates at a rate of 1E5 cells / well. Subsequently, Minimum Essential Medium Eagle (Sigma) containing 10% FBS was used as the culture medium for the HeLa cells. The following day, each well was replaced with fresh medium. Then, plasmid DNA encoding L7Ae (L7Ae-G4S-TagBFP, SEQ ID NO: 105), fused to TagBFP via a GS4 linker, was transfected into each well at a rate of 500 ng / well. Lipofectamine® 3000 (Invitrogen) was used as the transfection reagent.

[0239] Two days after plasmid DNA transfection, each well was again replaced with fresh medium. Subsequently, each well was transfected with 500 ng of each EGFP mRNA and 500 ng of iRFP670 mRNA (SEQ ID NO: 5) using Lipofectamine® MessengerMAX (Invitrogen).

[0240] The day after mRNA transfection, the culture medium was removed from each well, and each well was washed with PBS, followed by 0.1 mL of Accumax. TM Nacalai tesque was added to detach the cells. Then, 0.4 mL of culture medium was added and the cells were suspended. After centrifugation at 200 × g for 1 minute, the supernatant was removed and 0.5 mL of FACS buffer was added. The FACS buffer was prepared by adding 75 mL of MACS (registered trademark) BSA Stock Solution (Miltenyi Biotec) to 1.45 L of autoMACS (registered trademark) Rinsing Solution (Miltenyi Biotec). The FACS sample was prepared by centrifugation at 200 × g for 1 minute, removal of the supernatant, and addition of 0.5 mL of FACS buffer.

[0241] As a control, HeLa cells that had not been transfected with plasmid DNA or mRNA were seeded on the same day, and procedures other than transfection were performed to prepare FACS samples (untreated HeLa cells).

[0242] Subsequently, FACS measurements were performed using a Cell Sorter SH800 (SONY). Fluorescence was measured for TagBFP (405 nm laser, 450 / 50 filter) to assess L7Ae protein levels, EGFP (488 nm laser, 530 / 30 filter) to assess EGFP protein levels, and iRFP670 (640 nm laser, 670 / 30 filter) to assess mRNA transfection levels. After measurement, the fluorescence values ​​were analyzed as follows: First, cell fractions showing iRFP670 fluorescence (i.e., fractions of cells transfected with mRNA) were named RFP+ and gating was established. For cells included in RFP+, gating was set up on the TagBFP histogram based on the fluorescence intensity of TagBFP. Cell fractions with high TagBFP fluorescence intensity were named BFP high, those with low TagBFP fluorescence intensity were named BFP low, and those where the TagBFP fluorescence intensity overlapped with untreated cells were named BFP(-) (Figure 1). The median fluorescence values ​​of EGFP and iRFP670 were calculated for each cell fraction. Subsequently, the median EGFP fluorescence value for each cell fraction was divided by the median iRFP670 fluorescence value to calculate the value. This value is denoted as "EGFP / iRFP fluorescence intensity". Cell fractions labeled BFP(-) are recognized as having no L7Ae protein expression, BFP low cell fractions as having low L7Ae protein expression, and BFP high cell fractions as having high L7Ae protein expression. Furthermore, for each mRNA construct standardized in this way, the relative values ​​(normalized EGFP fluorescence) were calculated for the EGFP / iRFP fluorescence intensity of Control EGFP in each cell fraction of BFP(-), BFP low, and BFP high, with the EGFP / iRFP fluorescence intensity of Control EGFP set to 100.Even if it is not a control EGFP, if the EGFP mRNA construct is not degraded by miRNA and does not contain an aptamer domain, the normalized EGFP fluorescence intensity of the target EGFP mRNA construct can be calculated by setting the EGFP / iRFP fluorescence intensity using the said EGFP mRNA construct to 100. Furthermore, the L7Ae-dependent increase rate of EGFP translation (ON / OFF ratio) was calculated by dividing the normalized EGFP fluorescence intensity in the BFP-high cell fraction by the normalized EGFP fluorescence intensity in the BFP-- cell fraction.

[0243] The results are shown in Figures 3 and 4. Compared to Control EGFP, the normalized EGFP fluorescence intensity of EGFP_Tg21 was reduced to about 1 / 20, confirming that the presence of miR21 in HeLa cells causes translational repression of EGFP_Tg21 mRNA. On the other hand, in cells introduced with Control EGFP and cells introduced with EGFP_Tg21, no significant changes in normalized EGFP fluorescence intensity were observed with respect to the presence or absence of L7Ae protein expression or the amount of L7Ae protein expression, and no changes in translation amount were observed with respect to the presence or absence of L7Ae protein expression or the amount of L7Ae protein expression. On the other hand, in cells introduced with EGFP_CDmini_Tg21, a tendency for normalized EGFP fluorescence intensity to increase in an L7Ae-dependent manner was observed, and a tendency for the amount of EGFP translation to increase in an L7Ae-dependent manner was observed.

[0244] [Example 3] Evaluation of L7Ae-dependent increase in translation amount when a CDmini with the stem removed from the 5' or 3' side of the miR21 binding sequence is provided, and an antisense sequence is provided for the miR binding sequence. The L7Ae binding sequence used in this study (CDmini, SEQ ID NO: 112, SEQ ID NO: 211 for the sequence without U substitution with a modified nucleotide) [Citation: Saito H et al., Nature Chemical Biology volume 6, pages 71-78 (2010)] has three bases at each end that are hybridized, and there is a stem region consisting of three base pairs that can hybridize. The sequence with this stem region removed is called CDminiΔstem (SEQ ID NO: 113, SEQ ID NO: 212 for the sequence without U substitution with a modified nucleotide) (Figure 6).

[0245] As shown in Figure 6, control EGFP (SEQ ID NO: 6), EGFP_Tg21 (SEQ ID NO: 7), EGFP-encoded mRNA with a CDminiΔstem on the 5' side of the miR21 binding sequence and an antisense sequence for 3–9 bases from the 5' side of the miR21 binding sequence (EGFP_AS3~9_CD_Tg21, the number after AS represents the number of antisense bases, SEQ ID NOs: 9–14), and EGFP-encoded mRNA with a CDboxΔstem on the 3' side of the miR21 binding sequence and an antisense sequence for 3–8 bases from the 3' side of the miR21 binding sequence (EGFP_Tg21_CD_AS3~8, the number after AS represents the number of antisense bases, SEQ ID NOs: 15–20) were prepared, and experiments were conducted using HeLa cells (ATCC, Cat.No. CCL-2) to investigate the changes in translation levels of these constructs in the presence of L7Ae. The experimental procedures and analysis of the results were carried out in the same manner as in Example 2. BD LSRFortessa as a FACS device TM X-20 was used. The FACS measurement results and the gating settings for BFP(-), BFP low, and BFP high are shown in Figure 5.

[0246] The results are shown in Figures 7 and 8. It was confirmed that by adding an antisense sequence to the miR21 binding sequence, the normalized EGFP fluorescence intensity increased more clearly in a protein expression level-dependent manner than when the antisense sequence was not added, and the amount of EGFP translated increased in a protein expression level-dependent manner than when the antisense sequence was not added.

[0247] [Example 4] Evaluation of L7Ae-dependent increase in translation volume when a CDminiΔstem and an antisense sequence for the shRNA target sequence are provided on the 5' or 3' side of the shRNA target sequence, and shRNA is co-introduced.

[0248] As shown in Figure 10, EGFP-encoded mRNA (G_TgSh, SEQ ID NO: 21) with an shRNA-binding sequence (SEQ ID NO: 109) in the 3'UTR, EGFP-encoded mRNA (G_AS4~6_CD_TgSh, where the number after AS represents the number of antisense bases, SEQ ID NOs: 22~24) with a CDminiΔstem on the 5' side of the shRNA-binding sequence and an antisense sequence targeting 4 to 6 bases from the 5' side of the shRNA-binding sequence, and EGFP-encoded mRNA (G_TgSh_CD_AS6, where the number after AS represents the number of antisense bases, SEQ ID NO: 25) with a CDminiΔstem on the 3' side of the shRNA-binding sequence and an antisense sequence targeting 6 bases from the 3' side of the shRNA-binding sequence were created, and experiments were conducted using HeLa cells (ATCC, Cat.No. CCL-2) to investigate the changes in translation levels in the presence of L7Ae under conditions with and without co-introduction of shRNA (SEQ ID NO: 110).

[0249] The experimental procedures and analysis of results were carried out in the same manner as in Example 2. However, the transfection amounts for EGFP mRNA and iRFP670 mRNA were 200 ng / well, and shRNA was also transfected simultaneously with mRNA, with a shRNA transfection amount of 3 pmol / well. When co-transfecting mRNA and shRNA, the predetermined amounts of mRNA and shRNA were first mixed, and then mixed with Lipofectamine® messengerMAX (Invitrogen) to prepare the transfection solution. The shRNA synthesis was carried out using the in vitro Transcription T7 Kit (for siRNA Synthesis) (Takara) as follows: Forward primer (SEQ ID NO: 3) and reverse primer (SEQ ID NO: 4), which serve as templates for shRNA synthesis, were prepared in annealing buffer to a final concentration of 10 μM. The mixture was then heated in a thermal cycler at 95°C for 2 minutes and slowly cooled at room temperature. Subsequently, in vitro transcription was performed under the conditions described in the kit manual. Subsequently, shRNA was purified using the Monarch® RNA Cleanup Kit (New England Biolabs). Molar concentration calculations were performed using a molecular weight of 17284.8 for shRNA (SEQ ID NO: 110). A BD LSRFortessa FACS instrument was used. TM X-20 was used. When calculating the normalized EGFP fluorescence intensity, the EGFP / iRFP fluorescence intensity of G_TgSh (SEQ ID NO: 21) under conditions where shRNA (SEQ ID NO: 110) was not co-introduced was set to 100. The FACS measurement results and the gating settings for BFP(-), BFP low, and BFP high are shown in Figure 9.

[0250] The results are shown in Figures 11 and 12. Translation suppression was observed when shRNA was co-introduced into EGFP mRNA containing the shRNA target sequence (SEQ ID NO: 21). Furthermore, EGFP mRNA (SEQ ID NOs: 22-25) with a CDminiΔstem at the 5' or 3' end of the shRNA target sequence and an antisense sequence at 4-6 bases from the 5' or 3' end of the shRNA target sequence showed a stronger L7Ae-dependent increase in translation compared to mRNA without these antisense sequences (SEQ ID NO: 21).

[0251] [Example 5] Evaluation of L7Ae-dependent increase in translation when a CDminiΔstem is provided on the 5' or 3' side of the let7a binding sequence and an antisense sequence for the miR binding sequence is provided. As shown in Figure 14, EGFP encoded mRNA with a let7a binding sequence (G_TgLet7a, SEQ ID NO: 27), EGFP encoded mRNA with a shuffled sequence of the let7a binding sequence (a sequence that cannot bind to let7a) (G_TgLet7a_shuffle, SEQ ID NO: 26), EGFP encoded mRNA with a CDminiΔstem provided on the 5' side of the let7a binding sequence (G_CD_TgLet7a, SEQ ID NO: 28), and further, 3 to 7 from the 5' side of the let7a binding sequence. EGFP-encoded mRNAs were created with antisense sequences against specific bases (G_AS4~7_CD_TgLet7a, where the number after AS indicates the number of antisense bases, SEQ ID NOs: 29-31), EGFP-encoded mRNA with a CDminiΔstem on the 3' side of the let7a binding sequence (G_TgLet7a_CD, SEQ ID NO: 32), and further EGFP-encoded mRNA with antisense sequences against 3-7 bases from the 3' side of the let7a binding sequence (G_TgLet7a_CD_AS3~7, where the number after AS indicates the number of antisense bases, SEQ ID NOs: 33-36). Experiments were then conducted using HeLa cells (ATCC, Cat.No. CCL-2) to investigate the changes in translation levels of these constructs in the presence of L7Ae.

[0252] The experimental procedures and analysis of results were carried out in the same manner as in Example 2. However, the mRNA transfection amounts for EGFP and iRFP670 were each set to 250 ng, and a BD LSRFortessa was used as the FACS instrument. TM X-20 was used. The FACS measurement results and the gating settings for BFP(-), BFP low, and BFP high are shown in Figure 13. When calculating the normalized EGFP fluorescence intensity, the EGFP / iRFP fluorescence intensity of G_TgLet7a_shuffle was set to 100.

[0253] The results are shown in Figures 15 and 16. The construct containing the CDminiΔstem (SEQ ID NO: 28) showed a stronger L7Ae-dependent increase in normalized EGFP fluorescence intensity compared to the construct without the CDminiΔstem (SEQ ID NO: 27). Furthermore, it was confirmed that the constructs containing an antisense sequence against the let7a binding sequence (SEQ ID NOs: 29-31, 34, 35) showed an even more pronounced L7Ae-dependent increase in normalized EGFP fluorescence intensity.

[0254] [Example 6] Evaluation of MCP-dependent increase in translation when an MS2 aptamer is provided on the 5' or 3' side of the let7a binding sequence and an antisense sequence is provided for the miR binding sequence. As shown in Figure 18, mRNAs were created using the MS2 aptamer (MS2, SEQ ID NO: 114, SEQ ID NO: 213 for sequences where U is not substituted with a modified nucleotide) that binds to the MS2 coat protein (MCP, SEQ ID NO: 106) (Citation: Keryer-Bibens C. et al., Biol Cell. 2008 Feb;100(2):125-38.) or its stem removed (MS2Δstem (AGGAUCACC (SEQ ID NO: 214), a sequence in which U is substituted with N1-methyl-pseuduridine (m1ψ)), SEQ ID NO: 115) as shown in the figure, and experiments were conducted using HeLa cells (ATCC, Cat.No. CCL-2) to investigate the fluctuations in EGFP translation levels in the presence of MCP for these constructs.

[0255] The experimental procedure and analysis of results were carried out in the same manner as in Example 2. However, instead of L7Ae-G4S-TagBFP, a plasmid encoding MCP fused to TagBFP via a G4S linker (TagBFP-G4S-MCP, SEQ ID NO: 106) was transfected. The mRNA transfection amounts for EGFP and iRFP670 were each 250 ng, and a BD LSRFortessa FACS instrument was used. TM X-20 was used. The FACS measurement results and the gating settings for BFP(-), BFP low, and BFP high are shown in Figure 17. When calculating the normalized EGFP fluorescence intensity, the EGFP / iRFP fluorescence intensity of G_TgLet7a_shuffle was set to 100.

[0256] The results are shown in Figures 19 and 20. By adding the MS2 aptamer to the 5' end of the let7a binding sequence, a stronger MCP-dependent increase in EGFP translation was observed compared to the case without the MS2 aptamer. Furthermore, when a sequence with the MS2 aptamer stem removed was added, along with an antisense sequence for the let7a binding sequence, the MCP-dependent increase in EGFP translation was even more pronounced, regardless of whether the aptamer was placed on the 5' or 3' end of the miR binding sequence.

[0257] [Example 7] Evaluation of L7Ae-dependent increase in translation when a CDminiΔstem is provided on the 5' side of the let7a binding sequence and antisense sequences are provided for various sites of the miR binding sequence. mRNAs shown in Figure 22 were prepared, and experiments were conducted using HeLa cells (ATCC, Cat. No. CCL-2) to investigate the change in translation levels of these constructs in the presence of L7Ae. The experimental procedure and analysis of results were carried out in the same manner as in Example 2. However, the mRNA transfection amounts for EGFP and iRFP670 were each set to 250 ng, and a BD LSRFortessa was used as the FACS instrument. TMX-20 was used. The FACS measurement results and the gating settings for BFP(-), BFP low, and BFP high are shown in Figure 21. When calculating the normalized EGFP fluorescence intensity, the EGFP / iRFP fluorescence intensity of G_TgLet7a_shuffle was set to 100.

[0258] The results are shown in Figures 23 and 24. It was confirmed that even when nucleotides of the miRNA target sequence capable of forming a stem at the end of the aptamer domain are located inside the miRNA, the amount of EGFP translation increases in a way that is dependent on the expression level of L7Ae.

[0259] [Example 8] Evaluation of L7Ae-dependent increase in translation volume of mRNA with CDminiΔstem and antisense sequence for the miR binding sequence adjacent to the miR binding sequence of the let7 family. The same experiment as in Example 5 was performed on mRNA with miR binding sequences (SEQ ID NOs: 116-122) for let7 family members other than let7a (let7x: let7b, let7c, let7d, let7e, let7f, let7g, let7i). EGFP-encoded mRNA with a let7a binding sequence (G_TgLet7a, SEQ ID NO: 27), EGFP-encoded mRNA with a shuffled let7a binding sequence (a sequence that cannot bind to let7a) (G_TgLet7a_shuffle, SEQ ID NO: 26), EGFP-encoded mRNA with a let7x binding sequence (G_TgLet7x, SEQ ID NOs: 69, 74, 79, 83, 87, 91, 95), EGFP-encoded mRNA with a CDminiΔstem on the 3' side of the let7x binding sequence (G_CD_TgLet7x, SEQ ID NOs: 70, 75, 80, 84, 88, 92, 96), and further EGFP-encoded mRNA with an antisense sequence for 3-6 bases from the 3' side of the let7x binding sequence (G_AS3-6_CD_TgLet7x, the number after AS represents the number of bases in the antisense, SEQ ID NOs: 71-73, 76-78, Experiments to investigate the changes in translation levels in the presence of L7Ae were conducted using HeLa cells (ATCC, Cat.No. CCL-2) for cells 81, 82, 85, 86, 89, 90, 93, 94, and 97-99).

[0260] The configurations of G_TgLet7a and G_TgLet7a_shuffle are shown in Figure 14. G_TgLet7b contains the sequence encoding EGFP in its ORF and the let7b target sequence in its 3'UTR. G_TgLet7c contains the sequence encoding EGFP in its ORF and the let7c target sequence in its 3'UTR. G_TgLet7d contains the sequence encoding EGFP in its ORF and the let7d target sequence in its 3'UTR. G_TgLet7e contains the sequence encoding EGFP in its ORF and the let7e target sequence in its 3'UTR. G_TgLet7f contains the sequence encoding EGFP in its ORF and the let7f target sequence in its 3'UTR. G_TgLet7g contains the sequence encoding EGFP in its ORF and the let7g target sequence in its 3'UTR. G_TgLet7i contains the sequence encoding EGFP in its ORF and the let7i target sequence in its 3'UTR.

[0261] G_CD_TgLet7b contains an EGFP-coding sequence in its ORF, with the CDminiΔstem and let7b target sequence in the 3'UTR, where the CDminiΔstem and let7b target sequence are adjacent, and the CDminiΔstem and let7b target sequence are arranged in the order from 5' to 3'. G_CD_TgLet7c contains an EGFP-coding sequence in its ORF, with the CDminiΔstem and let7c target sequence in the 3'UTR, where the CDminiΔstem and let7c target sequence are adjacent, and the CDminiΔstem and let7c target sequence are arranged in the order from 5' to 3'. G_CD_TgLet7d contains an EGFP-coding sequence in its ORF, with the CDminiΔstem and let7d target sequence in the 3'UTR, where the CDminiΔstem and let7d target sequence are adjacent, and the CDminiΔstem and let7d target sequence are arranged in the order from 5' to 3'. G_CD_TgLet7e contains an EGFP-coding sequence in its ORF, with the CDminiΔstem and let7e target sequence in the 3'UTR, where the CDminiΔstem and let7e target sequence are adjacent, and the CDminiΔstem and let7e target sequence are arranged in the order from 5' to 3'. G_CD_TgLet7f contains an EGFP-coding sequence in its ORF, with the CDminiΔstem and let7f target sequence in the 3'UTR, where the CDminiΔstem and let7f target sequence are adjacent, and the CDminiΔstem and let7f target sequence are arranged in the order from 5' to 3'. G_CD_TgLet7g contains an EGFP-coding sequence in its ORF, with the CDminiΔstem and let7g target sequence in the 3'UTR, where the CDminiΔstem and let7g target sequence are adjacent, and the CDminiΔstem and let7g target sequence are arranged in the order from 5' to 3'. G_CD_TgLet7i contains the sequence encoding EGFP in its ORF, and includes the CDminiΔstem and let7i target sequence in its 3'UTR. The CDminiΔstem and let7i target sequence are adjacent to each other, and are arranged in the order from 5' to 3'.

[0262] Each G_ASn_CD_TgLet7b (where n is an integer from 3 to 5) contains an EGFP-coding sequence in its ORF, a let7b target sequence and an aptamer domain in its 3'UTR, the n nucleotides at the 3' end of the aptamer domain are also the n nucleotides at the 5' end of the let7b target sequence, the n nucleotides at the 5' end of the aptamer domain have a sequence complementary to the n nucleotides at the 3' end of the aptamer domain and are capable of forming a stem, and the portion of the aptamer domain other than the stem-forming nucleotides is the CDminiΔstem. Each G_ASn_CD_TgLet7c (where n is 3, 4, or 6) contains an EGFP-coding sequence in its ORF, a let7c target sequence and an aptamer domain in its 3'UTR, the n nucleotides at the 3' end of the aptamer domain are also the n nucleotides at the 5' end of the let7c target sequence, the n nucleotides at the 5' end of the aptamer domain have a sequence complementary to the n nucleotides at the 3' end of the aptamer domain and are capable of forming a stem, and the portion of the aptamer domain other than the stem-forming nucleotides is the CDminiΔstem. Each G_ASn_CD_TgLet7d (where n is 4 or 6) contains an EGFP-coding sequence in its ORF, a let7d target sequence and an aptamer domain in its 3'UTR, the n nucleotides at the 3' end of the aptamer domain are also the n nucleotides at the 5' end of the let7d target sequence, the n nucleotides at the 5' end of the aptamer domain have a sequence complementary to the n nucleotides at the 3' end of the aptamer domain and are capable of forming a stem, and the portion of the aptamer domain other than the stem-forming nucleotides is the CDminiΔstem.Each G_ASn_CD_TgLet7e (where n is 4 or 6) contains an EGFP-coding sequence in its ORF, a let7e target sequence and an aptamer domain in its 3'UTR, the n nucleotides at the 3' end of the aptamer domain are also the n nucleotides at the 5' end of the Let7e target sequence, the n nucleotides at the 5' end of the aptamer domain have a sequence complementary to the n nucleotides at the 3' end of the aptamer domain and are capable of forming a stem, and the portion of the aptamer domain other than the stem-forming nucleotides is the CDminiΔstem. Each G_ASn_CD_TgLet7f (where n is 4 or 6) contains an EGFP-coding sequence in its ORF, a let7f target sequence and an aptamer domain in its 3'UTR, the n nucleotides at the 3' end of the aptamer domain are also the n nucleotides at the 5' end of the let7f target sequence, the n nucleotides at the 5' end of the aptamer domain have a sequence complementary to the n nucleotides at the 3' end of the aptamer domain and are capable of forming a stem, and the portion of the aptamer domain other than the stem-forming nucleotides is the CDminiΔstem. Each G_ASn_CD_TgLet7g (where n is 4 or 6) contains an EGFP-coding sequence in its ORF, a let7g target sequence and an aptamer domain in its 3'UTR, the n nucleotides at the 3' end of the aptamer domain are also the n nucleotides at the 5' end of the let7g target sequence, the n nucleotides at the 5' end of the aptamer domain have a sequence complementary to the n nucleotides at the 3' end of the aptamer domain and are capable of forming a stem, and the portion of the aptamer domain other than the stem-forming nucleotides is the CDminiΔstem. Each G_ASn_CD_TgLet7i (where n is an integer from 3 to 5) contains an EGFP-coding sequence in its ORF, a let7i target sequence and an aptamer domain in its 3'UTR, the n nucleotides at the 3' end of the aptamer domain are also the n nucleotides at the 5' end of the let7i target sequence, the n nucleotides at the 5' end of the aptamer domain have a sequence complementary to the n nucleotides at the 3' end of the aptamer domain and are capable of forming a stem, and the portion of the aptamer domain other than the stem-forming nucleotides is the CDminiΔstem.

[0263] The experimental procedures and analysis of results were carried out in the same manner as in Example 2. However, the mRNA transfection amounts for EGFP and iRFP670 were each set to 250 ng, and a BD LSRFortessa was used as the FACS instrument. TM X-20 was used. The FACS measurement results and the gating settings for BFP(-), BFP low, and BFP high are shown in Figure 25. When calculating the normalized EGFP fluorescence intensity, the EGFP / iRFP fluorescence intensity of G_TgLet7a_shuffle was set to 100.

[0264] The results are shown in Figures 26 and 27. The inclusion of the L7Ae binding sequence (aptamer domain) resulted in a stronger, L7Ae-dependent increase in EGFP translation compared to the case without the L7Ae binding sequence. Furthermore, it was confirmed that the inclusion of antisense sequences against the binding sequences of each let7 family's miRs led to an even more pronounced L7Ae-dependent increase in EGFP translation.

[0265] [Example 9] Evaluation of L7AE-dependent increase in translation volume of mRNA with CDMINIΔSTEM and antisense sequence for MIR binding sequence adjacent to the MIR23A binding sequence. EGFP encoded mRNA with Let7a binding sequence (G_TgLet7a, SEQ ID NO: 27), EGFP encoded mRNA with a shuffled Let7a binding sequence (sequence that cannot bind to let7a) (G_TgLet7a_shuffle, SEQ ID NO: 26), EGFP encoded mRNA with miR23a binding site (G_TgmiR23a, SEQ ID NO: 100), EGFP encoded mRNA with CDminiΔstem on the 3' side of the miR23a binding sequence (G_CD_TgmiR23a, SEQ ID NO: 101), and further miR23a Experiments were conducted using HeLa cells (ATCC, Cat.No. CCL-2) to investigate the changes in translation levels of EGFP-encoded mRNA (G_AS3-6_CD_TgmiR23a, where the number after AS represents the number of bases in the antisense sequence, sequence numbers: 102-104) that had an antisense sequence attached to 3-6 bases from the 3' end of the binding sequence, in the presence of L7Ae.

[0266] The configurations of G_TgLet7a and G_TgLet7a_shuffle are shown in Figure 14. G_TgmiR23a contains the sequence encoding EGFP in its ORF and the miR23a target sequence in its 3'UTR. G_CD_TgmiR23a contains the sequence encoding EGFP in its ORF and the CDminiΔstem and miR23a target sequence in its 3'UTR, with the CDminiΔstem and miR23a target sequence being adjacent to each other, and the CDminiΔstem and miR23a target sequence being arranged in the order from 5' to 3'. Each G_ASn_CD_TgmiR23a (where n is 3, 4, or 6) contains an EGFP-encoding sequence in its ORF, and includes a miR23a target sequence and an aptamer domain in its 3'UTR, where the n nucleotides at the 3' end of the aptamer domain are also the n nucleotides at the 5' end of the miR23a target sequence, and the n nucleotides at the 5' end of the aptamer domain have a sequence complementary to the n nucleotides at the 3' end of the aptamer domain, allowing for stem formation, and the portion of the aptamer domain other than the stem-forming nucleotides is the CDminiΔstem.

[0267] The experimental procedures and analysis of results were carried out in the same manner as in Example 2. However, the mRNA transfection amounts for EGFP and iRFP670 were each set to 250 ng, and a BD LSRFortessa was used as the FACS instrument. TM X-20 was used. The FACS measurement results and the gating settings for BFP(-), BFP low, and BFP high are shown in Figure 28. When calculating the normalized EGFP fluorescence intensity, the EGFP / iRFP fluorescence intensity of G_TgLet7a_shuffle was set to 100.

[0268] The results are shown in Figures 29 and 30. The presence of the L7Ae binding sequence tended to increase EGFP translation in an L7Ae-dependent manner compared to the case without the L7Ae binding sequence. Furthermore, it was confirmed that the addition of an antisense sequence to the miR23a binding sequence resulted in a more pronounced L7Ae-dependent increase in EGFP translation.

[0269] [Example 10] Evaluation of L7Ae-dependent increase in translation by fusing an aptamer to the cytoplasmic side of a membrane protein. An experiment was conducted using the membrane protein CAXII (Carbonic anhydrase XII) with the aim of investigating whether the translation rate increases by fusing an aptamer to the cytoplasmic portion of a membrane protein. CAXII is a single-pass transmembrane protein, and it has been reported that the N-terminus faces the extracellular side and the C-terminus faces the cytoplasm [Citation: Tafreshi N. et al., Subcell Biochem. 2014:75:221-54.]. CAXII-TagBFP (SEQ ID NO: 124), which has TagBFP fused to the C-terminus of CAXII, was expressed in cultured cells and confirmed to be expressed on the cell membrane (data not shown). We investigated whether an increase in translation rate similar to that in Example 5 could be observed by further fusing L7Ae to the C-terminus of CAXII-TagBFP.

[0270] Experiments were conducted using HeLa cells (ATCC, Cat.No. CCL-2) to investigate the changes in translation levels of EGFP-encoded mRNA in the presence of CAXII-TagBFP-L7Ae (Sequence ID: 107) for EGFP-encoded mRNA with a let7a binding sequence (G_TgLet7a, SEQ ID: 27), EGFP-encoded mRNA with a shuffled let7a binding sequence (a sequence that cannot bind to let7a) (G_TgLet7a_shuffle, SEQ ID: 26), and EGFP-encoded mRNA with a CDminiΔstem on the 5' side of the let7a binding sequence and an antisense sequence for 4 bases from the 5' side of the let7a binding sequence (G_AS4_CD_TgLet7a_2, SEQ ID: 54).

[0271] The structures of G_TgLet7a and G_TgLet7a_shuffle are shown in Figure 14. G_AS4_CD_TgLet7a_2 contains the sequence encoding EGFP in its ORF, and the 3'UTR contains the let7a target sequence and an aptamer domain. The four nucleotides at the 3' end of the aptamer domain are also the four nucleotides at the 5' end of the let7a target sequence, and the four nucleotides at the 5' end of the aptamer domain have a sequence complementary to the four nucleotides at the 3' end of the aptamer domain, allowing them to form a stem. The portion of the aptamer domain other than the stem-forming nucleotides is the CDminiΔstem.

[0272] The experimental procedure and analysis of results were carried out in the same manner as in Example 2. However, instead of L7Ae-G4S-TagBFP, a plasmid encoding CAXII-TagBFP-L7Ae (SEQ ID NO: 107) was transfected. In addition, the mRNA transfection amounts for EGFP and iRFP670 were each 250 ng, and a BD LSRFortessa was used as the FACS instrument. TM X-20 was used. The FACS measurement results and the gating settings for BFP(-), BFP low, and BFP high are shown in Figure 31. When calculating the normalized EGFP fluorescence intensity, the EGFP / iRFP fluorescence intensity of G_TgLet7a_shuffle was set to 100.

[0273] The results are shown in Figures 32 and 33. By providing antisense sequences for the L7Ae binding sequence and the let7a binding sequence, an increase in the amount of EGFP translation dependent on CAXII-TagBFP-L7Ae was confirmed.

[0274] [Example 11] Evaluation of L7Ae or MCP-dependent increase in translation when a CDmini or MS2 aptamer is provided at 5' of the let7a binding sequence and an antisense sequence is provided against the miR binding sequence. Experiments were conducted using HeLa cells (ATCC, Cat.No. CCL-2) to investigate the changes in EGFP translation levels in the presence of L7Ae or MCP for mRNA using CDmini, CDminiΔstem, MS2, and MS2Δstem.

[0275] EGFP-encoded mRNA with a let7a binding sequence (G_TgLet7a, SEQ ID NO: 27), EGFP-encoded mRNA with a shuffled let7a binding sequence (a sequence that cannot bind to let7a) (G_TgLet7a_shuffle, SEQ ID NO: 26), EGFP-encoded mRNA with CDmini on the 5' side of the let7a binding sequence (G_delA3_CDmini_TgLet7a, SEQ ID NO: 55), or EGFP-encoded mRNA with CDminiΔstem on the 5' side (G_delA3 EGFP-encoded mRNA with CD_TgLet7a (SEQ ID NO: 56), or an antisense sequence attached to the 5' end of the CDminiΔstem and let7a binding sequence for 3-7 bases from the 5' end (G_delA3_AS3_CD_TgLet7a~G_delA3_AS7_CD_TgLet7a, SEQ ID NO: 57~61), EGFP-encoded mRNA with MS2 attached to the 5' end (G_delA3_MS2_TgLet7a, SEQ ID NO: 62), EGFP-encoded mRNA with MS2Δstem attached to the 5' end (G_delA3_MS2Δstem_TgLet7a, SEQ ID NO: 63), EGFP-encoded mRNA with an antisense sequence attached to the 5' end of the MS2Δstem and let7a binding sequence for 3-7 bases from the 5' end (G_delA3_AS3_MS2Δstem_TgLet7a~G_delA3_AS7_ Experiments were conducted using HeLa cells (ATCC, Cat.No. CCL-2) to investigate the changes in translation levels of MS2Δstem_TgLet7a (sequence numbers: 64-68) in the presence of L7Ae-TagBFP (sequence number: 105) or TagBFP-MCP (sequence number: 106).

[0276] The configurations of G_TgLet7a and G_TgLet7a_shuffle are shown in Figure 14. G_delA3_CDmini_TgLet7a contains the sequence encoding EGFP in its ORF, the let7a target sequence and CDmini in its 3'UTR, and the CDmini and let7a target sequence are arranged sequentially from the 5' end.

[0277] G_delA3 CD_TgLet7a contains the sequence encoding EGFP in its ORF, with the let7a target sequence and CDminiΔstem in the 3'UTR, and the CDminiΔstem and let7a target sequence arranged sequentially from the 5' end.

[0278] G_delA3_ASn_CD_TgLet7a (where n is an integer from 3 to 7) contains an EGFP-coding sequence in its ORF, and its 3'UTR contains the let7a target sequence and an aptamer domain. The n nucleotides at the 3' end of the aptamer domain are also the n nucleotides at the 5' end of the let7a target sequence, and the n nucleotides at the 5' end of the aptamer domain have a sequence complementary to the n nucleotides at the 3' end of the aptamer domain, allowing them to form a stem. The portion of the aptamer domain other than the stem-forming nucleotides is the CDminiΔstem.

[0279] G_delA3_MS2_TgLet7a contains the sequence encoding EGFP in its ORF, the let7a target sequence and MS2 in its 3'UTR, and MS2 and the let7a target sequence are arranged sequentially from the 5' end. G_delA3_MS2Δstem_TgLet7a contains the sequence encoding EGFP in its ORF, the let7a target sequence and MS2Δstem in its 3'UTR, and MS2Δstem and the let7a target sequence are arranged sequentially from the 5' end.

[0280] G_delA3_ASn_MS2Δstem_TgLet7a (where n is an integer from 3 to 7) contains an EGFP-coding sequence in its ORF, a let7a target sequence and an aptamer domain in its 3'UTR, the n nucleotides at the 3' end of the aptamer domain are also the n nucleotides at the 5' end of the let7a target sequence, the n nucleotides at the 5' end of the aptamer domain have a sequence complementary to the n nucleotides at the 3' end of the aptamer domain and are capable of forming a stem, and the portion of the aptamer domain other than the stem-forming nucleotides is the MS2Δstem.

[0281] The experimental procedures and analysis of results were carried out in the same manner as in Example 2. However, the mRNA transfection amounts for EGFP and iRFP670 were each set to 250 ng, and a BD LSRFortessa was used as the FACS instrument. TM X-20 was used. Furthermore, when preparing FACS samples, 50 μL of culture medium was added to cells detached with Accumax (Nacalai tesque), and this was used as the FACS sample. The FACS measurement results and the gating settings for BFP(-), BFP low, and BFP high are shown in Figure 34. When calculating normalized EGFP fluorescence intensity, the EGFP / iRFP fluorescence intensity of G_TgLet7a_shuffle was set to 100.

[0282] The results are shown in Figures 35, 36, 37, and 38. The presence of L7Ae or MCP binding sequences resulted in a stronger, L7Ae or MCP-dependent increase in EGFP translation compared to the case without these sequences. Furthermore, the inclusion of an antisense sequence against the let7a binding sequence further enhanced the L7Ae or MCP-dependent increase in EGFP translation.

[0283] [Example 12] Evaluation of MCP-dependent increase in translation volume of mRNA with an MS2Δstem and an antisense sequence for the miR binding sequence located adjacent to the miR122 and miR126 binding sequences. The same experiment as in Example 5 was performed on mRNA with a miR binding sequence (sequence number: 232) for miR122 (sequence number: 141). EGFP-encoded mRNA with a shuffled let7a binding sequence (a sequence that cannot bind to let7a) (G_TgLet7a_shuffle, SEQ ID NO: 26), EGFP-encoded mRNA with a miR122 binding sequence (G_Tg122, SEQ ID NO: 237), EGFP-encoded mRNA with an MS2Δstem on the 3' side of the miR122 binding sequence (G_Tg122_MS2Post, SEQ ID NO: 238), EGFP-encoded mRNA with an antisense sequence at 4 or 5 bases from the 3' side of the miR122 binding sequence (G_Tg122_MS2Post_AS4-5, the number after AS represents the number of bases in the antisense, SEQ ID NOs: 239, 240), and an MS2Δstem on the 5' side of the miR122 binding sequence Experiments were conducted using HeLa cells (ATCC, Cat.No. CCL-2) to investigate the changes in EGFP translation levels in the presence of MCP for EGFP-encoded mRNA with an antisense sequence (G_MS2Pre_Tg122, SEQ ID NO: 241) and EGFP-encoded mRNA with an antisense sequence added to 3-5 bases from the 5' end of the miR122 binding sequence (G_AS3-5_MS2Pre_Tg122, SEQ ID NO: 242-244).

[0284] The same experiment as in Example 5 was performed on mRNA containing a miR binding sequence (sequence number: 233) for miR126 (sequence number: 142). EGFP encoded mRNA containing a shuffled let7a binding sequence (a sequence that cannot bind to let7a) (G_TgLet7a_shuffle, sequence number: 26), EGFP encoded mRNA containing a miR126 binding sequence (G_Tg126, sequence number: 245), EGFP encoded mRNA containing an MS2Δstem on the 3' side of the miR126 binding sequence (G_Tg126_MS2Post, sequence number: 246), EGFP encoded mRNA containing an antisense sequence at 3 or 5 bases from the 3' side of the miR126 binding sequence (G_Tg126_MS2Post_AS3,5, the number after AS represents the number of bases in the antisense, sequence numbers: 247, 248), and MS2Δstem on the 5' side of the miR126 binding sequence Experiments were conducted using HeLa cells (ATCC, Cat.No. CCL-2) to investigate the changes in EGFP translation levels in the presence of MCP for EGFP-encoded mRNA with an antisense sequence (G_MS2Pre_Tg126, SEQ ID NO: 249) and EGFP-encoded mRNA with an antisense sequence attached to 3 or 6 bases from the 5' end of the miR126 binding sequence (G_AS3,6_MS2Pre_Tg126, SEQ ID NO: 250, 251).

[0285] The configuration of G_TgLet7a_shuffle is shown in Figure 14. G_Tg122 contains the sequence encoding EGFP in its ORF and the miR122 target sequence in its 3'UTR. G_Tg126 contains the sequence encoding EGFP in its ORF and the miR126 target sequence in its 3'UTR.

[0286] G_Tg122_MS2Post contains an EGFP-coding sequence in its ORF, with the miR122 target sequence and MS2Δstem in the 3'UTR, where the miR122 target sequence and MS2Δstem are adjacent, and the miR122 target sequence and MS2Δstem are arranged in the order from 5' to 3'. G_Tg126_MS2Post contains an EGFP-coding sequence in its ORF, with the miR126 target sequence and MS2Δstem in the 3'UTR, where the miR126 target sequence and MS2Δstem are adjacent, and the miR126 target sequence and MS2Δstem are arranged in the order from 5' to 3'.

[0287] Each G_Tg122_MS2Post_ASn (where n is 4 or 5) contains an EGFP-coding sequence in its ORF, a miR122 target sequence and an MS2Δstem in its 3'UTR, the n nucleotides at the 5' end of the aptamer domain are also the n nucleotides at the 3' end of the miR122 target sequence, the n nucleotides at the 5' end of the aptamer domain have a sequence complementary to the n nucleotides at the 3' end of the aptamer domain and are capable of forming a stem, and the portion of the aptamer domain other than the stem-forming nucleotides is the MS2Δstem. Each G_Tg126_MS2Post_ASn (where n is 3 or 5) contains an EGFP-coding sequence in its ORF, a miR126 target sequence and an MS2Δstem in its 3'UTR, the n nucleotides at the 5' end of the aptamer domain are also the n nucleotides at the 3' end of the miR126 target sequence, the n nucleotides at the 5' end of the aptamer domain have a sequence complementary to the n nucleotides at the 3' end of the aptamer domain and are capable of forming a stem, and the portion of the aptamer domain other than the stem-forming nucleotides is the MS2Δstem.

[0288] G_MS2Pre_Tg122 contains an EGFP-coding sequence in its ORF, with the MS2Δstem and miR122 target sequence in its 3'UTR, where the MS2Δstem and miR122 target sequence are adjacent, and the MS2Δstem and miR122 target sequence are arranged in the order from 5' to 3'. G_MS2Pre_Tg126 contains an EGFP-coding sequence in its ORF, with the MS2Δstem and miR126 target sequence in its 3'UTR, where the MS2Δstem and miR126 target sequence are adjacent, and the MS2Δstem and miR126 target sequence are arranged in the order from 5' to 3'.

[0289] Each G_ASn_MS2Pre_Tg122 (where n is an integer from 3 to 5) contains an EGFP-coding sequence in its ORF, an MS2Δstem miR122 target sequence in its 3'UTR, the n nucleotides at the 3' end of the aptamer domain are also the n nucleotides at the 5' end of the miR122 target sequence, the n nucleotides at the 5' end of the aptamer domain have a sequence complementary to the n nucleotides at the 3' end of the aptamer domain and are capable of forming a stem, and the portion of the aptamer domain other than the stem-forming nucleotides is the MS2Δstem. Each G_ASn_MS2Pre_Tg126 (where n is 3 or 6) contains an EGFP-coding sequence in its ORF, an MS2Δstem miR126 target sequence in its 3'UTR, the n nucleotides at the 3' end of the aptamer domain are also the n nucleotides at the 5' end of the miR126 target sequence, the n nucleotides at the 5' end of the aptamer domain have a sequence complementary to the n nucleotides at the 3' end of the aptamer domain and are capable of forming a stem, and the portion of the aptamer domain other than the stem-forming nucleotides is the MS2Δstem.

[0290] The experimental procedure and analysis of results were carried out in the same manner as in Example 2. First, HeLa cells were seeded in a 96-well plate at a rate of 1E4 cells / well. In addition, instead of L7Ae-G4S-TagBFP, a plasmid encoding MCP fused to TagBFP via a linker (TagBFP-G4S-MCP-2, SEQ ID NO: 261) was transfected into each well at a rate of 0.1 μg / well. EGFP and iRFP670 mRNA were also transfected into each well at a rate of 20 ng / well each, and simultaneously, mirVana was used as a miRNA mimic reagent. TM miRNA mimic (Invitrogen, 4464066, miR122 mimic: Assay ID MC11012, miR126 mimic: Assay ID 12841) was also transfected into each well at 0.1 pmol / well. When co-transfecting mRNA and miRNA, predetermined amounts of mRNA and miRNA were first mixed, then mixed with Lipofectamine® messengerMAX (Invitrogen) to prepare the transfection solution. Additionally, mirVana was used as a miRNA mimic negative control. TM miRNA Mimic, Negative Control #1 (Invitrogen, 4464058) was used.

[0291] The day after mRNA transfection, the culture medium was removed from each well, and each well was washed with PBS, followed by 0.05 mL of Accumax. TM (Nacalai tesque) was added and the cells were detached. Then, 0.1 mL of culture medium was added and the cells were suspended. A BD LSRFortessa was used as the FACS instrument. TM The X-20 was used.

[0292] The FACS measurement results and the gating settings for BFP(-), BFP low, and BFP high are shown in Figure 39. When calculating the normalized EGFP fluorescence intensity, the EGFP / iRFP fluorescence intensity when G_TgLet7a_shuffle and miRNA mimic negative control were co-administered was set to 100.

[0293] The results are shown in Figures 40 (40-1, 40-2) and 41 (41-1, 41-2). The inclusion of an MCP-binding sequence (aptamer domain) resulted in a tendency for EGFP translation to increase in an MCP-dependent manner compared to the case without the MCP. Furthermore, it was confirmed that the inclusion of an antisense sequence for each miR binding sequence led to a more pronounced MCP-dependent increase in EGFP translation.

[0294] [Example 13] Evaluation of MCP-dependent increase in translation volume of mRNA with a CS2Δstem and an antisense sequence for the miR binding sequence located adjacent to the miR122 and miR142 binding sequences. The same experiment as in Example 5 was performed on mRNA with a miR binding sequence (Sequence ID: 232) for miR122. EGFP-encoded mRNA with a shuffled let7a binding sequence (a sequence that cannot bind to let7a) (G_TgLet7a_shuffle, SEQ ID NO: 26; SEQ ID NO: 275 for the sequence without U substitution with a modified nucleotide), EGFP-encoded mRNA with a miR122 binding sequence (G_Tg122, SEQ ID NO: 237), and a CS2 aptamer (CS2, SEQ ID NO: 235) that binds to the L7Ae variant LS12 on the 3' side of the miR122 binding sequence (Citation: Fukunaga K. et al., Nucleic Acids Res. 2021 Jul 5;50(2):601-616.) with the stem removed (CS2Δstem, SEQ ID NO: 236). Experiments were conducted using HeLa cells (ATCC, Cat.No. CCL-2) to investigate the changes in EGFP translation levels in the presence of LS12 for EGFP-encoding mRNA with an antisense sequence (G_Tg122_CS2, SEQ ID NO: 252) and EGFP-encoding mRNA with an antisense sequence attached to 4-6 bases from the 3' end of the miR122 binding sequence (G_Tg122_CS2_AS4-6, the number after AS represents the number of bases in the antisense sequence, SEQ ID NO: 253-255).

[0295] The same experiment as in Example 5 was performed on mRNA containing a miR binding sequence (sequence number: 234) for miR142 (sequence number: 144). EGFP encoded mRNA containing a shuffled let7a binding sequence (a sequence that cannot bind to let7a) (G_TgLet7a_shuffle, sequence number: 26), EGFP encoded mRNA containing a miR142 binding sequence (G_Tg142, sequence number: 256), EGFP encoded mRNA containing a CS2 aptamer (CS2, sequence number: 235) with the stem removed (CS2Δstem, sequence number: 236) that binds to the L7Ae variant LS12 (CS2, sequence number: 235) on the 3' side of the miR142 binding sequence, and EGFP encoded mRNA containing an antisense sequence for 4-6 bases from the 3' side of the miR142 binding sequence (G_Tg142_CS2_AS4-6, Experiments were conducted using HeLa cells (ATCC, Cat.No. CCL-2) to investigate the changes in EGFP translation levels in the presence of LS12 for sequence numbers 258-260 (the number after AS represents the antisense base number).

[0296] The configuration of G_TgLet7a_shuffle is shown in Figure 14. G_Tg122 contains the sequence encoding EGFP in its ORF and the miR122 target sequence in its 3'UTR. G_Tg142 contains the sequence encoding EGFP in its ORF and the miR142 target sequence in its 3'UTR.

[0297] G_Tg122_CS2 contains an EGFP-coding sequence in its ORF, with the miR122 target sequence and CS2Δstem in the 3'UTR, where the miR122 target sequence and CS2Δstem are adjacent, and the miR122 target sequence and CS2Δstem are arranged in the order from 5' to 3'. G_Tg142_CS2 contains an EGFP-coding sequence in its ORF, with the miR142 target sequence and CS2Δstem in the 3'UTR, where the miR142 target sequence and CS2Δstem are adjacent, and the miR142 target sequence and CS2Δstem are arranged in the order from 5' to 3'.

[0298] Each G_Tg122_CS2_ASn (where n is an integer from 4 to 6) contains an EGFP-coding sequence in its ORF, a miR122 target sequence and a CS2Δstem in its 3'UTR, the n nucleotides at the 5' end of the aptamer domain are also the n nucleotides at the 3' end of the miR122 target sequence, the n nucleotides at the 5' end of the aptamer domain have a sequence complementary to the n nucleotides at the 3' end of the aptamer domain and are capable of forming a stem, and the portion of the aptamer domain other than the stem-forming nucleotides is the CS2Δstem. Each G_Tg142_CS2_ASn (where n is an integer from 4 to 6) contains an EGFP-coding sequence in its ORF, a miR142 target sequence and a CS2Δstem in its 3'UTR, the n nucleotides at the 5' end of the aptamer domain are also the n nucleotides at the 3' end of the miR142 target sequence, the n nucleotides at the 5' end of the aptamer domain have a sequence complementary to the n nucleotides at the 3' end of the aptamer domain and are capable of forming a stem, and the portion of the aptamer domain other than the stem-forming nucleotides is the CS2Δstem.

[0299] The experimental procedure and analysis of results were carried out in the same manner as in Example 2. First, HeLa cells were seeded in a 96-well plate at a rate of 1E4 cells / well. In addition, instead of L7Ae-G4S-TagBFP, a plasmid encoding LS12 (LS12-G4S-TagBFP, SEQ ID NO: 262) fused to TagBFP via a linker was transfected into each well at a rate of 33 ng / well. EGFP and iRFP670 mRNA were also transfected into each well at a rate of 18 ng / well each, and simultaneously, mirVana was used as a miRNA mimic reagent. TMmiRNA Mimic (Invitrogen, 4464066, miR122: Assay ID MC11012, miR142: Assay ID 10398) was also transfected into each well at 1 pmol / well. When co-transfecting mRNA and miRNA, predetermined amounts of mRNA and miRNA were first mixed, then mixed with Lipofectamine® messengerMAX (Invitrogen) to prepare the transfection solution. Additionally, mirVana was used as a miRNA mimic negative control. TM miRNA Mimic, Negative Control #1 (Invitrogen, 4464058) was used.

[0300] The day after mRNA transfection, the culture medium was removed from each well, and each well was washed with PBS, followed by 0.05 mL of Accumax. TM (Nacalai tesque) was added and the cells were detached. Then, 0.1 mL of culture medium was added and the cells were suspended. A BD LSRFortessa was used as the FACS instrument. TM The X-20 was used.

[0301] The FACS measurement results and the gating settings for BFP(-), BFP low, and BFP high are shown in Figure 42. When calculating the normalized EGFP fluorescence intensity, the EGFP / iRFP fluorescence intensity when co-transfected with G_TgLet7a_shuffle and miRNA mimic negative control was set to 100.

[0302] The results are shown in Figures 43 (Figures 43-1 and 43-2) and 44 (Figures 44-1 and 44-2). The presence of an LS12 binding sequence (aptamer domain) resulted in a tendency for EGFP translation to increase in an LS12-dependent manner compared to the case without the LS12 binding sequence. Furthermore, it was confirmed that the addition of antisense sequences to the binding sequences of each miR resulted in a more pronounced LS12-dependent increase in EGFP translation.

[0303] [Example 14] Synthesis of circular RNA Each circular RNA was prepared according to Wesselhoeft, RA et al, Nat Commun 9, 2629 (2018) and Kameda, S. et al, Nucleic Acids Research, Volume 51, Issue 4, 28 February 2023, Page e24. The basic structure of the plasmid encoding each RNA before circularization is shown in Figure 45. As the circularization module, a PIE (Permuted Intron-exon) of an intron and exon derived from Anabaena pre-tRNA, which is a Group I intron, was used. In addition, to improve the circularization efficiency, mutually complementary external homology arm (EHA) and internal homology arm (IHA) sequences were introduced according to the aforementioned literature. As the IRES (Internal ribosome entry site) element for initiating translation, an IRES derived from Coxackievirus B3 (CVB3) was used, and the sequence encoding eGFP was placed downstream thereof. As in previous examples, the 5'UTR and 3'UTR regions were appropriately inserted with a miRNA Let7a target sequence (Let7a site) and an MCP binding sequence (MS2 motif). The constructed construct is shown in Table 2, and the structure of the circular RNA synthesized from the constructed construct by the method described later is shown in Figure 46. The 5'UTR and 3'UTR in the constructed construct refer to the 5'UTR and 3'UTR in the linear RNA translated from the Construct DNA.

[0304]

[0305] Plasmids containing the template DNA sequences for each mRNA were cleaved with NotI-HF (NEB, R3189) and then purified using the QIAquick® PCR Purification Kit (Qiagen). The purified DNA was then used to create MEGAscript TMRNA was synthesized using the T7 Transcription Kit (Invitrogen). Subsequently, DNase I (New England Biolabs, M0303L) was added to degrade the DNA in the reaction mixture, and the mixture was incubated at 37°C for 20 minutes. After that, the RNA was purified using the RNeasy® mini kit (Qiagen).

[0306] Circularization was performed using various prepared linear RNAs. Each RNA was heated at 75°C for 5 minutes and then allowed to stand on ice for at least 2 minutes. Subsequently, GTP and MgCl2 were added to each RNA to final concentrations of 2 mM and 10 mM, respectively, and the RNA was incubated at 55°C for 15 minutes. After that, the RNA was extracted using the RNeasy® mini kit (Qiagen). To remove linear RNA, RNaseR (Lucigen, RNR07250) was further added, and the reaction was carried out according to the protocol provided with the reagents. After the reaction, the RNA was extracted using the RNeasy® mini kit (Qiagen). The extracted RNA was subjected to confirmation electrophoresis using E-gel EX 2%. ssRNA ladder (NEB, N0362S) was used as a marker. The results are shown in Figure 47. In all samples, two bands were observed, similar to the references mentioned above: a main band representing circular RNA (top) and linear RNA with nicks (bottom).

[0307] [Example 15] Evaluation of MCP-dependent translational increase when circular RNA is provided with a let7a binding sequence and an MS2 aptamer. Using the circular RNA shown in Figure 46 (circular RNA synthesized using the procedure of Example 14 with DNA sequences shown in SEQ ID NOs: 223-231 as templates), the translational increase in HeLa cells constitutively expressing MCP was evaluated.

[0308] HeLa cells constitutively expressing MCP were prepared using the following procedure. Plasmid DNA encoding the neomycin resistance gene and TagBFP-G4S-MCP-2 (SEQ ID NO: 261) was transfected into HeLa cells (ATCC, Cat. No. CCL-2) using Lipofectamine® 3000 (Invitrogen). After being cultured for 16 days in a medium containing Geneticin® Selective Antibiotic (Gibco), single cell sorting was performed using a Cell Sorter SH800 (SONY). Further culture was then performed to obtain cell lines. FACS measurements were performed on the obtained lines in the same manner as in Example 12, and HeLa cells with low MCP expression (hereinafter referred to as HeLa-MCP-low cells) and HeLa cells with high MCP expression (hereinafter referred to as HeLa-MCP-high cells) were identified based on the fluorescence intensity of TagBFP. Furthermore, the expression level of TagBFP-G4S-MCP-2 in these cell lines is less than one-quarter of the expression level of TagBFP-MCP in Example 6.

[0309] HeLa cells, HeLa-MCP-low cells, and HeLa-MCP-high cells were seeded in 96-well plates at a rate of 1E4 cells / well. The culture medium for HeLa cells was the same composition as in Example 2. The culture medium for HeLa-MCP-low cells and HeLa-MCP-high cells contained 1 mg / mL Geneticine (Gibco), and the rest of the composition was the same as in Example 2. The following day, each well was replaced with fresh medium. Each well was transfected with 19 ng each of circular RNA (circular RNA synthesized using the procedure of Example 14 with DNA sequences shown in SEQ ID NOs: 223-231 as templates) and 19 ng each of iRFP670 mRNA (SEQ ID NO: 5) using Lipofectamine® MessengerMAX (Invitrogen).

[0310] FACS measurements and results analysis were performed in the same manner as in Example 12. However, a Novocyte Quanteon (Agilent) was used as the FACS instrument. For gating, the cell fraction in which iRFP670 fluorescence was observed (i.e., the fraction of cells transfected with mRNA) was named RFP+ and set as such. For HeLa cells, HeLa-MCP-low cells, and HeLa-MCP-high cells, the normalized EGFP fluorescence intensity was calculated, with the EGFP / iRFP fluorescence intensity of CircRNA (circular RNA synthesized using the procedure in Example 14 with the DNA sequence shown in SEQ ID NO: 223 as a template) in the RFP+ fraction set to 100. Furthermore, the MCP-dependent increase rate of EGFP translation (ON / OFF ratio) was calculated by dividing the normalized EGFP fluorescence intensity in HeLa-MCP-high cells by the normalized EGFP fluorescence intensity in HeLa cells.

[0311] The results are shown in Figures 48 and 49. When the let7a binding sequence was included in only one of the 5'UTR or 3'UTR, it was confirmed that the amount of EGFP translation increased in an MCP-dependent manner by providing an MS2Δstem on the 3' side of the let7a binding sequence and an antisense sequence against the let7a binding sequence. When the let7a binding sequence was provided in both the 5'UTR and 3'UTR, and only one of the let7a binding sequences in either the 5'UTR or 3'UTR had an MS2Δstem and an antisense sequence against the let7a binding sequence on the 3' side, the MCP-dependent increase in translation was slight. On the other hand, when the let7a binding sequence was provided in both the 5'UTR and 3'UTR, and both the let7a binding sequences in the 5'UTR and 3'UTR had an MS2Δstem and an antisense sequence against the let7a binding sequence on the 3' side, the MCP-dependent increase in translation was greater.

[0312] [Example 16] Evaluation of MCP-dependent translational increase when a let7a binding sequence and MS2 aptamer are provided on the 5'UTR As shown in Figure 50, MCP-dependent translational increase was evaluated using G_TgLet7a_shuffle (SEQ ID NO: 26) or G_TgLet7a (SEQ ID NO: 27), RNA (SEQ ID NOs: 263-271) with a let7a binding sequence, MS2Δstem, and an antisense sequence for the let7a binding sequence on the 5'UTR.

[0313] Evaluation was performed using HeLa cells and HeLa-MCP cells prepared in Example 15. Cell culture and mRNA transfection were carried out in the same manner as in Example 15. FACS measurement and result analysis were performed in the same manner as in Example 12. However, for gating, the cell fraction in which iRFP670 fluorescence was observed (i.e., the fraction of cells transfected with mRNA) was named RFP+ and set as such. For HeLa cells, HeLa-MCP-low cells, and HeLa-MCP-high cells, the normalized EGFP fluorescence intensity was calculated with the EGFP / iRFP fluorescence intensity of G_TgLet7a_shuffle in the RFP+ fraction set to 100. Furthermore, the MCP-dependent increase rate of EGFP translation (ON / OFF ratio) was calculated by dividing the normalized EGFP fluorescence intensity in HeLa-MCP-high cells by the normalized EGFP fluorescence intensity in HeLa cells. The results are shown in Figures 51 and 52. It was confirmed that the amount of EGFP translation increased in an MCP-dependent manner by adding the MS2Δstem and an antisense sequence for the let7a binding sequence to the 3' end of the let7a binding sequence.

[0314] [Example 17] Evaluation of MCP-dependent translation increase when plasmid DNA encoding a construct having an MS2 aptamer on the 3' side of the let7a binding sequence and an antisense sequence for the miR binding sequence is introduced into cells. Using plasmid DNA encoding the G_TgLet7a_MS2Δstem_AS4 (SEQ ID NO: 44, however, 1-methylpseudridine is replaced with uridine) construct shown in Figure 18, an experiment was conducted using HeLa cells (ATCC, Cat.No. CCL-2) to investigate the change in EGFP translation levels in the presence of TagBFP-G4S-MCP-2 (SEQ ID NO: 261).

[0315] HeLa cells were cultured in the same manner as in Example 2. 101 ng of plasmid DNA encoding either G_TgLet7a_shuffle (SEQ ID NO: 26, with 1-methylpseudridine replaced by uridine), G_TgLet7a (SEQ ID NO: 27, with 1-methylpseudridine replaced by uridine), or G_TgLet7a_MS2Δstem_AS4 (SEQ ID NO: 44, with 1-methylpseudridine replaced by uridine) was mixed with 101 ng of plasmid DNA encoding iRFP670 (SEQ ID NO: 5) and 101 ng of plasmid DNA encoding TagBFP-G4S-MCP-2 (SEQ ID NO: 261). As a control, a sample without the TagBFP-MCP-2-encoding plasmid DNA was also prepared. The mixed plasmid DNAs were transfected into HeLa cells using Lipofectamine® 3000 (Invitrogen).

[0316] Two days after plasmid DNA transfection, FACS measurement and analysis of the results were performed in the same manner as in Example 2. However, a BD LSRFortessa was used as the FACS instrument. TM X-20 was used. Additionally, during cell detachment, the culture medium was removed from each well, and each well was washed with PBS before adding 0.1 mL of Accumax. TMNacalai tesque was added. Subsequently, 0.4 mL of culture medium was added and the cells were suspended. For gating, the cell fraction in which iRFP670 fluorescence was observed (i.e., the fraction of cells transfected with mRNA) was named RFP+ and set as such. Furthermore, for both conditions with and without the addition of plasmid DNA encoding TagBFP-G4S-MCP-2, the normalized EGFP fluorescence intensity was calculated, with the EGFP / iRFP fluorescence intensity of G_TgLet7a_shuffle in the RFP+ fraction set to 100. In addition, the MCP-dependent increase rate of EGFP translation (ON / OFF ratio) was calculated by dividing the normalized EGFP fluorescence intensity under the condition with the addition of plasmid DNA encoding TagBFP-G4S-MCP-2 by the normalized EGFP fluorescence intensity under the condition without the addition of plasmid DNA encoding TagBFP-G4S-MCP-2.

[0317] The results are shown in Figures 53 and 54. It was confirmed that when an MS2Δstem was provided on the 3' side of the let7a binding sequence, and an antisense sequence was provided for the let7a binding sequence, the amount of EGFP translation increased in an MCP-dependent manner.

Claims

1. An RNA molecule comprising an open reading frame (ORF) containing a sequence encoding a target protein, an aptamer domain capable of binding to a protein different from the target protein (target protein), and a short single-stranded nucleic acid target domain containing a target sequence to which a short single-stranded nucleic acid can bind, wherein the number of nucleotides in the short single-stranded nucleic acid is 200 or less.

2. The RNA molecule according to claim 1, wherein the aptamer domain binds to the target protein, thereby inhibiting the binding of the target sequence to the short single-stranded nucleic acid.

3. The RNA molecule according to claim 1 or 2, wherein the short single-stranded nucleic acid target domain is included in the aptamer domain, the short single-stranded nucleic acid target domain is adjacent to the aptamer domain, one or more nucleotides in the aptamer domain are included as part of the target sequence of the short single-stranded nucleic acid target domain, or one or more nucleotides in the target sequence of the short single-stranded nucleic acid target domain are included as part of the aptamer domain.

4. The RNA molecule according to any one of claims 1 to 3, wherein the sequence located at the 5' end and the sequence located at the 3' end of the aptamer domain are capable of forming a stem.

5. The RNA molecule according to any one of claims 1 to 4, wherein one or more nucleotides located at the 5' or 3' end of the aptamer domain constitute a part of the target sequence.

6. The RNA molecule according to any one of claims 1 to 5, wherein the aptamer domain and the short single-stranded nucleic acid target domain are directly linked without overlap.

7. The RNA molecule according to any one of claims 1 to 6, wherein the short single-stranded nucleic acid capable of binding to the target sequence is an RNA having one of sequence numbers: 125-134, 141, 142, or 144.

8. The RNA molecule according to any one of claims 1 to 6, wherein the short single-stranded nucleic acid capable of binding to a target sequence is endogenous RNA or exogenous RNA.

9. The RNA molecule according to any one of claims 1 to 8, wherein the portion other than the sequence encoding the target protein is an untranslated region (UTR).

10. The RNA molecule according to any one of claims 1 to 9, wherein the translation of a target protein is inhibited by the binding of a short single-stranded nucleic acid to the target sequence of the short single-stranded nucleic acid target domain.

11. A DNA molecule having a sequence that, upon transcription, yields the RNA molecule described in any one of claims 1 to 10.

12. A composition comprising an RNA molecule according to any one of claims 1 to 10, or a DNA molecule according to claim 11.

13. The composition according to claim 12, further comprising a short single-chain nucleic acid or a short single-chain nucleic acid precursor, wherein the short single-chain nucleic acid or the short single-chain nucleic acid produced from the short single-chain nucleic acid precursor is capable of binding to the target sequence.

14. The composition according to claim 12 or 13 for use in the treatment or prevention of a disease.

15. A method for expressing a target protein in a cell, comprising introducing an RNA molecule according to any one of claims 1 to 10 or a DNA molecule according to claim 11 into a cell in which the target protein of the aptamer domain is present.

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