JP2012023998A - Method for controlling expression of target gene in eukaryotic cell, rna molecule and dna molecule - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide an RNA molecule for simply controlling expression of transgene by a desired molecule without using an RNA enzyme and an RNA except an mRNA, a DNA molecule encoding the RNA molecule, and a method for expressing a target gene in eukaryotic cells using the DNA molecule.SOLUTION: The RNA molecule is an RNA molecule for controlling expression of a target gene by a ligand, and has a specific 5' terminal stem loop, a modulator sequence, an anti-anti-inner ribosome binding site sequence, an aptamer sequence, an anti-inner ribosome binding site sequence, an inner ribosome binding site and a target gene from a 5' side.

Description

  The present invention relates to a method for controlling the expression of a target gene in a eukaryotic cell, and an RNA molecule and a DNA molecule used in the method.

  Cells have various gene expression regulation mechanisms, such as when a specific enzyme is needed, it begins to express the gene that encodes it, or a specific gene is overexpressed. I try not to.

  As such a mechanism, the lactose operon of E. coli is famous. The lactose operon is controlled so that β-galactosidase, which is a lactose degrading enzyme, is produced when glucose is not present and lactose is present.

  If this lactose operon is utilized, gene expression can be promoted by a lactose analog such as IPTG in prokaryotic cells. However, compounds used for promoting gene expression are limited to lactose analogs, and gene expression cannot be controlled by a desired molecule. In addition, this lactose operon cannot be used in eukaryotic cells.

  Molecular responsive gene expression control mechanisms that function in eukaryotic cells have also been found to a small extent. However, there are still limited compounds that can be used for expression control.

  In addition, mechanisms that control gene expression by specific molecules such as metabolites have been found in prokaryotes. Specifically, as shown in FIG. 1, in this mechanism, a ribosome binding site (RBS) exists upstream of a gene encoding a protein, and a binding site (aptamer) such as a metabolite exists further upstream. Normally, the ribosome binds to the ribosome binding site and the downstream gene is translated, but when a metabolite or the like binds to the aptamer, the structure of the ribosome binding site changes and the ribosome cannot bind, resulting in inhibition of translation. Is done. In this way, some prokaryotic cells have suppressed overexpression of specific genes. Such a mechanism is called a riboswitch because ribonucleic acid (RNA) controls gene expression by itself.

  Aptamers that bind to the desired molecule can be obtained by in vitro artificial evolution. Therefore, if a method for constructing an artificial riboswitch system using the aptamer can be established, gene expression can be controlled by the desired molecule. . In addition, riboswitches found in nature are usually of the type that suppresses gene expression as described above, but it may also be possible to construct a promotion type riboswitch that is more useful industrially. In fact, several artificial riboswitch systems that work in prokaryotic cells have been reported.

  However, the prokaryotic riboswitch cannot be applied to eukaryotic cells. In eukaryotic cells, unlike prokaryotic cells, translation is not controlled by whether or not ribosomes bind to ribosome binding sites inside mRNA, but ribosomes bind to the 5 ′ end of mRNA and translation begins. It depends.

  However, even in eukaryotes, a technique for controlling translation of mRNA by a desired molecule has been developed.

  For example, in Patent Document 1 and Non-Patent Documents 1 and 2, as shown in FIG. 2, an aptamer that selectively binds to a specific compound is inserted into the 5 ′ untranslated region or splicing site of mRNA, Disclosed is a system that inhibits scanning by ribosomes and splicing by spliceosomes by binding, and consequently inhibits translation. However, even though such a system can inhibit translation, it cannot promote translation using a specific molecule.

  Patent Document 2 and Non-Patent Documents 3 to 4 describe a system in which a nucleic acid (ribozyme) whose cleavage activity is suppressed by a specific molecule or a nucleic acid (aptozyme) whose cleavage activity is imparted by a specific molecule is inserted into mRNA. Is disclosed. The present inventors have developed a system for detecting a specific molecule by applying a similar system (Patent Document 3). However, such a system is complicated and difficult to design itself. Further, since it involves the cleavage of nucleic acid, it can only be controlled in one direction, and the responsiveness of the system greatly depends on the activity of the nucleic acid enzyme.

  Furthermore, Patent Documents 4 to 5 and Non-Patent Documents 5 to 7 describe systems in which molecular responsiveness is imparted to RNA different from mRNA, and translation is controlled by an antisense effect or RNAi effect. However, since this system requires RNA different from mRNA, it is necessary to adjust the transcription amount and transcription site of the two types of RNA. In addition, when a plurality of RNAs are used, there is a problem that the possibility of adversely affecting other genes increases.

US Patent Application Publication No. 2002/0006661 International Publication No. 00/24912 Pamphlet JP 2008-220191 A US Patent Application Publication No. 2006/0088864 US Patent Application Publication No. 2009/0143327

Geoffrey Werstuck et al., SCIENCE, 282, 296-298 (1998) Dong-Suk Kim et al., RNA, No. 11, pp. 1667-1677 (2005) Leising Yen et al., Nature, 431, 471-476 (2004) Maung Nyan Win et al., Proceedings of the National Academy of Sciences, 104, 36, 14283-14288 (2007) Travis S Bayer et al., Nature Biotechnology, 23, 3, 337-343 (2005) Chase L Beisel et al., Molecular Systems Biology, No. 4, pp. 1-14 (2008) Deepak Kumar et al., Journal of the American Chemical Society, No. 131, pp. 13906-13907 (2009)

  As described above, conventionally, systems for controlling transgene expression and translation in eukaryotic cells have already been developed. However, there has been no technique that can easily control the expression of a transgene with a desired molecule without using RNA or RNA enzyme other than mRNA.

  Therefore, the problem to be solved by the present invention is to provide an RNA molecule for easily controlling the expression of a transgene with a desired molecule without using RNA or RNA enzyme other than mRNA. Another object of the present invention is to provide a DNA molecule encoding the RNA molecule and a method for expressing the target gene in eukaryotic cells using the DNA molecule.

  This inventor repeated earnest research in order to solve the said subject. As a result, viral mRNA having an internal ribosome binding site sequence is used, and in particular, a 5 ′ end stem loop is introduced at its 5 ′ end to suppress major 5 ′ end dependent translation in eukaryotic cells, and By introducing appropriate modulator and aptamer sequences to control the change in the three-dimensional structure of the internal ribosome binding site in the presence or absence of the ligand, the expression of the target gene can be clarified according to the presence or absence of the ligand. As a result, the present invention was completed.

  The RNA molecule according to the present invention is an RNA molecule that can control the expression of a target gene by a ligand, and has the following structure and sequence from the 5 'side.

(A) a 5 ′ end stem loop that inhibits 5 ′ end dependent translation;
(B) a modulator sequence that forms a modulator sequence stem loop by complementary binding to a portion of the aptamer sequence described below in the absence of a ligand;
(C) When a ligand is absent, a stem loop is formed together with a modulator sequence and a part of the following aptamer sequence. When a ligand is present, the following anti-internal ribosome binding site sequence (hereinafter referred to as “IRES” (Internal Ribosome Entry) Site) and an anti-anti-IRES sequence forming a complementary duplex;
(D) an aptamer sequence capable of binding to a ligand;
(E) In the absence of a ligand, it forms a complementary duplex with a portion of the IRES to inhibit ribosome binding to the IRES, and in the presence of a ligand, it forms an complementary duplex with the anti-anti-IRES sequence. An IRES sequence;
(F) IRES; and (g) gene of interest

  The complementary base length of the anti-IRES sequence for the IRES is preferably 6 or more and 12 or less. If the complementary base length is 6 or more, the anti-IRES sequence more reliably forms a complementary strand with the IRES in the absence of a ligand, effectively inhibiting IRES-dependent translation by inhibiting the function of the IRES. Can be suppressed. If the complementary base length is 12 or less, the anti-IRES sequence can be more reliably detached from the IRES due to the presence of the ligand, and the IRES-dependent translation can be started sufficiently.

  The complementary base length of the anti-anti-IRES sequence relative to the anti-IRES sequence is preferably 6 or more and 12 or less. If the complementary base length is 6 or more, when a ligand is present, the anti-anti-IRES sequence more reliably forms a complementary strand with the anti-IRES sequence and inhibits the action of the anti-IRES sequence, It becomes possible to effectively proceed with IRES-dependent translation. If the complementary base length is 12 or less, the anti-IRES sequence can be detached from the anti-anti-IRES sequence in the absence of the ligand, and can be more reliably bound to the IRES and inactivated. .

  When the modulator sequence forms a complementary duplex with not only a part of the aptamer sequence but also a part of the anti-anti-IRES sequence, the complementary base length is preferably 5 or less. The base sequence of the portion that binds to a part of the anti-anti-IRES sequence of the modulator sequence must be the same as a part of the anti-IRES sequence. Therefore, if the complementary base length is excessively long, the modulator sequence itself may bind to IRES and inhibit IRES sequence-dependent translation. However, when the complementary base length is 5 or less, such inhibition can be effectively suppressed.

  The complementary base length of the modulator sequence for a part of the aptamer sequence is preferably 4 or more and 8 or less. When the complementary base length is 4 or more, the modulator sequence binds more reliably to the aptamer sequence when no ligand is present, and IRES-dependent translation can be effectively suppressed. On the other hand, when the complementary base length is 8 or less, the modulator sequence is more reliably detached from the aptamer sequence in accordance with the three-dimensional structural change of the aptamer due to the presence of the ligand, and the anti-anti-IRES sequence and the anti-IRES sequence. The binding to the sequence is not suppressed, and thus IRES-dependent translation can be promoted. That is, when the complementary base length is 4 or more and 8 or less, an on / off switch for IRES-dependent translation by a ligand works very effectively.

  However, the preferred complementary base length between a part of the aptamer sequence and the modulator sequence also depends on the aptamer sequence. Thus, it is preferable that the same preferred embodiment is defined by the stability of the modulator array stem loop structure. As such a criterion, the Gibbs free energy (ΔG) of the modulator array stem loop structure is preferably −17.5 kcal / mol or more and −8.5 kcal / mol or less. For the same reason as described above, the modulator sequence stem-loop structure due to the presence or absence of the ligand, that is, the binding and desorption of the modulator sequence and a part of the anti-anti-IRES sequence and a part of the aptamer sequence are clarified. The switch of IRES-dependent translation by ligands works very effectively.

  The above-mentioned IRES is preferably those of the Chabanus stink bug enteric virus, and the anti-IRES sequence is capable of binding to the pseudonodule III or the stem loop V in the three-dimensional structure of the IRES of the Chabae stink bug enteric virus. Is preferred. The excellent effects of these IRES and anti-IRES sequences have been demonstrated by experiments described below.

  The DNA molecule according to the present invention encodes the above RNA molecule, and has a promoter sequence upstream of the coding region.

  A first method for controlling the expression of a target gene according to the present invention in a eukaryotic cell includes a step of mixing and reacting the RNA molecule or DNA molecule with an eukaryotic cell extract. .

  In addition, a second method for controlling the expression of a target gene according to the present invention in a eukaryotic cell includes a step of transfecting the DNA molecule into a eukaryotic cell; and by introducing a ligand into the eukaryotic cell. And a step of activating an internal ribosome binding site.

  The above RNA molecule, DNA molecule and gene expression control method is very useful in that eukaryotic cells or extracts thereof can be used and the expression of the target gene can be clearly controlled depending on the presence or absence of the ligand.

  According to the present invention, conventionally, control of transgene expression in eukaryotic cells could only be controlled off unless a complex system or the like was used, whereas simple control using a viral IRES (internal ribosome binding site). This system enables on-regulation of transgene expression.

  Therefore, the present invention can control the expression of transgenes using ligands, produce proteins and other compounds such as drugs, treat and diagnose diseases, biosensors and bioimaging, synthetic biology, protein functions and cell activities It is very useful in industry as a wide range of applications such as exhaustive analysis. Moreover, since the nucleic acid according to the present invention can be expressed and translated in viruses, it can also be applied to virus research.

FIG. 1 is a diagram schematically showing a riboswitch in a prokaryotic cell. FIG. 2 is a diagram schematically showing an artificial riboswitch in a eukaryotic cell, that is, an artificial off-switch using an aptamer. FIG. 3 is a schematic diagram of the genome of the chaban blue stink bug enterovirus. FIG. 4 is a schematic diagram of the structures of five template mRNAs used in a preliminary experiment (Experimental Example 1) described later. FIG. 5 is a graph showing the results of a luciferase assay in a preliminary experiment (Experimental Example 1) described later. FIG. 6 is a diagram showing the three-dimensional structure of the IRES of the chaban blue stink bug enteric virus. FIG. 7 is a graph showing the results of a luciferase assay in a preliminary experiment (Experimental Example 2) described later, in which a three-dimensional structure important for the activity of IRES was searched. FIG. 8 is a graph showing the results of a luciferase assay in a preliminary experiment (Experimental Example 3) described below, in which the complementary base length of an anti-IRES sequence sufficient to suppress the activity of IRES is examined. FIG. 9 shows the results of a luciferase assay in a preliminary experiment (Experimental Example 4) to be described later, in which the complementary base length of the anti-anti-IRES sequence sufficient to inhibit the IRES inactivation effect of the anti-IRES sequence was examined. It is a graph. FIG. 10 is a schematic diagram showing a specific example of an RNA molecule according to the present invention. (1) shows the case where no ligand is present, that is, the case where IRES is not activated and IRES-dependent translation is not performed, and (2) shows the case where the ligand is present, ie, when IRES is activated and IRES is dependent The case where sexual translation has started is shown. FIG. 11 is a graph showing the results of the luciferase assay in Example 1 described later, in which the base length of the modulator sequence was examined in the RNA molecule according to the present invention. FIG. 12 is a diagram showing examples of ligands that can be used in the present invention and aptamer base sequences corresponding thereto. FIG. 13 is a graph showing the results of a luciferase assay in Example 2 described later using various ligands and aptamers thereof.

  Hereinafter, the structure and sequence of the RNA molecule according to the present invention will be described from the 5 'end side. A specific example of the RNA molecule according to the present invention is shown in FIG.

(A) 5 ′ terminal stem loop The 5 ′ terminal stem loop is present at the 5 ′ terminal of the RNA molecule according to the present invention, and has a stem loop structure in which the terminal sequences form complementary strands. Its main role is to inhibit 5 ′ end-dependent translation in eukaryotic cells, ie translation initiated by the binding of ribosomes to the 5 ′ end of mRNA. It also has a role of stabilizing the formation of a complementary strand between the modulator sequence and a part of the anti-anti-IRES sequence and a part of the aptamer sequence.

  The 5 ′ terminal stem loop is not particularly limited as long as it exhibits the above-mentioned effects, but the base length may be at least about 10 bases, preferably about 10 bases or more and about 200 bases or less. Preferably it is about 12 base length or more and about 100 base length or less, More preferably, it is about 15 base length or more and about 50 base length or less. The length of the complementary strand formed by the both terminal sequences is preferably about 4 to 45 base length, more preferably about 6 to 22 base length. The length of the loop part is preferably about 3 to 10 bases, more preferably about 4 to 5 bases. Alternatively, a naturally occurring 5'-end stem loop such as the 5 'end of the g10 leader sequence of T7 phage or an artificial synthetic product thereof may be used.

  In addition, the complementary strand in the present invention is not only a completely complementary strand consisting only of the combination of AU and GC, but also as a whole, even if there are other combinations between them, the melting as much as the complete complementary strand as a whole Incomplete complementary strands indicating temperature (Tm) are also included.

(B) Modulator sequence The modulator sequence is a sequence for increasing the control efficiency of IRES-dependent translation by the presence or absence of a ligand. More specifically, in the absence of a ligand, formation of a complementary loop between the anti-anti-IRES sequence and the anti-IRES sequence is suppressed by forming a stem-loop structure together with the anti-anti-IRES sequence and the aptamer sequence. Suppress translation. On the other hand, when a ligand is present, the anti-anti-IRES sequence and the anti-IRES sequence can be formed by desorption from the aptamer sequence according to the three-dimensional structural change of the aptamer due to the binding of the ligand to the aptamer. -Remove the IRES sequence from the IRES so that IRES dependent translation can be initiated.

  Since the modulator sequence is complementary to a part of the aptamer sequence, the base sequence at the 3 'end may be complementary to the 5' end of the aptamer sequence.

  The modulator sequence may form a complementary duplex with part of the anti-anti-IRES sequence. In that case, the base sequence at the 5 'end is complementary to the 3' end of the anti-anti-IRES sequence.

  What is necessary is just to optimize the base length of a modulator arrangement | sequence so that the said effect | action can be exhibited. For example, when a complementary duplex is formed between a modulator sequence and an anti-anti-IRES sequence, the base length of the complementary sequence is preferably 1 or more and 5 or less. Since the modulator sequence and the anti-anti-IRES sequence are complementary and further complementary to the anti-anti-IRES sequence and the anti-IRES sequence, a part of the modulator sequence is identical to the anti-IRES sequence. Therefore, if the complementary base length is excessively long, the modulator sequence itself may bind to IRES and inhibit IRES sequence-dependent translation. However, when the complementary base length is 5 or less, such inhibition can be effectively suppressed.

  Moreover, as a complementary base length with respect to a part of aptamer sequence of a modulator sequence, 4 or more and 8 or less are suitable. When the complementary base length is 4 or more, the modulator sequence binds more reliably to the aptamer sequence when no ligand is present, and IRES-dependent translation can be effectively suppressed. On the other hand, when the complementary base length is 8 or less, the modulator sequence is more reliably detached from the aptamer sequence in accordance with the three-dimensional structural change of the aptamer due to the presence of the ligand, and the anti-anti-IRES sequence and the anti-IRES sequence. The binding to the sequence is not suppressed, and thus IRES-dependent translation can be promoted. That is, when the complementary base length is 4 or more and 8 or less, the IRES-dependent translation switch by the ligand works very effectively.

  Furthermore, the modulator array can be designed very simply by calculating the Gibbs free energy (ΔG) of the modulator array stem-loop structure and making the value appropriate. More specifically, first, regarding an aptamer, if a ligand is determined as described later, a base sequence capable of binding selectively and firmly to the ligand can be determined by a conventional method. The anti-anti-IRES sequence can also be determined as its complementary strand by determining an anti-IRES sequence that can effectively inhibit the action of IRES. A modulator sequence capable of constituting a stem-loop structure with a part of the aptamer sequence determined in this way and a part of the anti-anti-IRES sequence is appropriately designed. For example, computer software such as RNA structure (Mathews, DH et al. , Proceedings of the National Academy of Sciences, No. 101, pages 7287-7292 (2004)), the Gibbs free energy (ΔG) of the stem-loop structure is calculated, and the value is preferably −17.5 kcal / mol. As described above, it may be set to −8.5 kcal / mol or less. If the value is −8.5 kcal / mol or less, the modulator sequence stem-loop structure can be maintained sufficiently stably when no ligand is present, and IRES-dependent translation can be more reliably suppressed. On the other hand, if the value is too small, the modulator sequence stem-loop structure may not be resolved even if a ligand is present, and IRES-dependent translation may not be started. Therefore, the value is −17.5 kcal / mol or more. preferable. Note that the Gibbs free energy (ΔG) by RNA structure can be calculated in a few seconds.

(C) anti-anti-IRES sequence The anti-anti-IRES sequence forms a modulator sequence and a modulator sequence stem loop when no ligand is present, and is complementary to the anti-IRES sequence when a ligand is present. An anti-IRES sequence is released from the IRES and activates the IRES.

  The base sequence of the anti-anti-IRES sequence is a sequence suitable for forming a stem loop with the modulator sequence. For example, when the 3 'end portion forms a complementary double strand with the 5' end portion of the modulator sequence and becomes the stem portion of the stem loop, both base sequences are complementary to each other. At the same time, when a ligand is present, a complementary double strand is formed with the anti-IRES sequence, so that it must be complementary to the anti-IRES sequence.

  The anti-anti-IRES sequence may be designed so as to sufficiently exhibit the above-described action, but can preferably be a complementary strand with the anti-IRES sequence. Here, the anti-anti-IRES sequence preferably has a base length complementary to the anti-IRES sequence of 6 or more. If the complementary base length is 6 or more, when a ligand is present, the anti-anti-IRES sequence more reliably forms a complementary strand with the anti-IRES sequence and inhibits the action of the anti-IRES sequence, It becomes possible to effectively proceed with IRES-dependent translation. On the other hand, if the complementary chain length is too long, the affinity with the anti-IRES sequence becomes excessive, and even when no ligand is present, the inactivation action of the IRES by the anti-IRES sequence is not sufficiently exhibited, and it depends on IRES. Since there is a possibility that sexual translation may be initiated, the complementary strand length is preferably 15 or less, and more preferably 12 or less.

(D) Aptamer An aptamer is capable of selectively and firmly binding to a ligand, and has an action of activating IRES by changing the three-dimensional structure due to the presence of the ligand. More specifically, a modulator sequence stem loop is formed with a modulator sequence and an anti-anti-IRES sequence in the absence of a ligand. On the other hand, when a ligand is present, its three-dimensional structure changes due to inclusion of the ligand, and adjacent anti-anti-IRES sequences and anti-IRES sequences are brought close to each other to form complementary duplexes. IRES is activated by desorption from and initiates IRES-dependent translation.

  The base sequence of the aptamer is referred to as an in vitro artificial evolution method (referred to as SELEX (Systematic Evolution of Ligands by Exponential Enrichment) method, see Tuerk, C. et al., Science, Vol. 240, pages 505 to 510 (1990)). Therefore, it is possible to optimize depending on the ligand to be used. In this method, an oligonucleotide library that is a mixture of a large number of oligonucleotides having random base sequences is allowed to act on a ligand, an oligonucleotide having a high affinity is selected, and the selected oligonucleotide is amplified. Is. This makes it possible to optimize oligonucleotides that bind selectively and tightly to the ligand.

(E) anti-IRES sequence The anti-IRES sequence has an action of activating or deactivating IRES by detaching from or binding to IRES depending on the presence or absence of a ligand. More specifically, in the absence of a ligand, the anti-IRES sequence binds to an important portion of the IRES structure and inactivates the IRES. On the other hand, when a ligand is present, the IRES is activated because it forms a complementary duplex with the anti-anti-IRES sequence and desorbs from the IRES.

  The anti-IRES sequence can be designed as a complementary strand to a portion important for exerting the effect of IRES. For example, important three-dimensional structures such as loops, stem loops, and false nodules are found by analyzing the base sequence of the IRES used or X-ray crystal structure analysis. On top of that, in addition to IRES, mRNA having a 5 'terminal stem loop, a reporter gene such as luciferase, and a complementary strand to a specific site of IRES, or a similar mRNA and a specific site of IRES except for not having the complementary strand In a preliminary experiment using antisense DNA or antisense RNA complementary to, a reporter gene expression test is performed to determine an important part for IRES-dependent translation. The anti-IRES sequence may be a base sequence complementary to the important part determined in this way.

  The base length complementary to IRES of the anti-IRES sequence is preferably 6 or more. If the complementary base length is 6 or more, the anti-IRES sequence more reliably forms a complementary strand with the IRES in the absence of a ligand, effectively inhibiting IRES-dependent translation by inhibiting the function of the IRES. Can be suppressed. On the other hand, if the complementary base length is too long, the anti-IRES sequence cannot be detached from the IRES even if a ligand is present, and the IRES may not be activated. Is preferable, and 12 or less is more preferable.

(F) IRES
The IRES (internal ribosome binding site) is mainly present inside the viral RNA molecule and is involved in the initiation of translation, and translation is initiated by binding of 40S ribosome or 60S ribosome here.

  The IRES may be used after determining its three-dimensional structure as described above and specifying a site important for exerting its action and effect. However, the IRES has already been clarified in its base sequence and three-dimensional structure. It can also be used. Examples of such IRES include IRES such as Chabanae stink bug enterovirus, cricket paralysis virus, hepatitis C virus, poliovirus and the like.

  According to the experimental findings of the present inventor, the three-dimensional structure of pseudo-nodule III and stem loop V is very important for IRES-dependent translation in the IRES of the Chabae stink bug enteric virus. The sequence may be complementary to the base sequence related to these three-dimensional structures.

(G) Target gene The target gene of the present invention may be any gene desired to be expressed by the nucleic acid molecule according to the present invention, and is not particularly limited. For example, a gene encoding a useful protein desired to be produced; a luciferase gene or a GFF gene (for diagnosis, biosensor, bioimaging, etc.); a gene encoding a protein whose role or network is to be clarified (protein analysis, etc.) A gene responsible for part of the metabolic cascade (for synthetic biology, etc.).

  The sequence at the 3 'end is not particularly limited as long as it does not inhibit the action and effect of the present invention, and need not be provided. For example, a sequence that binds to a target gene or IRES and inhibits its expression or action cannot be used as a matter of course. On the other hand, as a measure against exonuclease, it is also possible to provide a neutral sequence of 100 bases or more that does not cause a problem at the 3 'end. Furthermore, a poly A sequence may be introduced.

  The ligand used in the present invention is not particularly limited as long as it binds to an aptamer, changes its three-dimensional structure, and activates IRES in the RNA molecule according to the present invention. Can do. For example, a relatively low-molecular organic compound; an inorganic compound such as a metal ion; a protein such as a growth factor, an enzyme, a receptor, a membrane protein, or a viral protein and a part thereof can be used.

  For example, when a ligand is taken into a cell from the outside through a cell membrane, a water-soluble low molecular weight organic compound can be used. Examples of such a ligand include theophylline, flavin mononucleotide, tetracycline, and sulforhodamine. In addition, intracellular molecules can be used as ligands. However, it is preferable to use a ligand that does not show cytotoxicity.

  The DNA molecule according to the present invention encodes the above RNA molecule, and has a promoter sequence upstream of the coding region. Such a DNA molecule is useful in that a desired target gene can be expressed using a desired ligand in a gene expression system comprising a eukaryotic cell itself or an eukaryotic cell extract.

  More specifically, the target gene can be expressed by transfecting and expressing a DNA molecule according to the present invention in a eukaryotic cell, and further activating IRES by introducing a ligand into the cell. In addition, an eukaryotic cell extract containing a gene expression system and a DNA molecule according to the present invention can be mixed to promote transcription of the DNA into RNA and translation into a protein, thereby expressing a target gene.

  What is necessary is just to select suitably the promoter of the DNA molecule based on this invention according to the eukaryotic cell to be used. For example, a viral promoter such as GAL for yeast and SV40 for animals can be used.

  The DNA molecule transfection means according to the present invention is not particularly limited. Generally, the DNA molecule according to the present invention is introduced into a eukaryotic cell after being incorporated into a plasmid vector or viral vector according to the eukaryotic cell to be used. In addition, there is a possibility that homologous recombination can be achieved by providing homologous sequences of the DNA of the eukaryotic cell to be used at both ends of the DNA molecule according to the present invention.

  The RNA molecule and DNA molecule according to the present invention described above can be expressed in a target gene by adding a ligand after being introduced into a eukaryotic cell or virus. The target gene can also be expressed in vitro using an expression system. That is, in the present invention, expression of a target gene in a eukaryotic cell is not limited to an embodiment using the eukaryotic cell itself, but an embodiment in which the target gene is expressed in a cell-free manner using a gene expression system of a eukaryotic cell. Shall also be included.

  When using a gene expression system comprising an eukaryotic cell extract, specifically, first, an eukaryotic cell extract containing a substance for synthesizing a protein from DNA or RNA, such as ribosome, is prepared. For example, such an extract from plant cells can be obtained by pulverizing plant seeds to select germs, washing the germs, then finely pulverizing them, and extracting with methanol or ethanol. Moreover, you may use a commercial item for this extract. Next, the obtained eukaryotic cell extract and the DNA or RNA may be mixed and reacted. At that time, a substrate necessary for protein synthesis such as amino acid may be added, or a substance necessary for transcription from DNA to RNA such as RNA polymerase may be further added. The reaction temperature and reaction time may be appropriately adjusted. For example, the reaction can be performed at 20 ° C. or more and 40 ° C. or less for 30 minutes or more and 10 hours or less.

  EXAMPLES Hereinafter, the present invention will be described more specifically with reference to examples. However, the present invention is not limited by the following examples, but may be appropriately modified within a range that can meet the purpose described above and below. Of course, it is possible to implement them, and they are all included in the technical scope of the present invention.

Experimental Example 1 Optimization of PSIV IRES (1) Preparation of template DNA In the genomic RNA (Fig. 3) of Chabaen stink bug enterovirus (hereinafter referred to as "PSIV"), it has 5951-6204 bases and is a nonstructural protein precursor 53 bases of the 3 'end of the first ORF that encodes the 5' end, 5 'end of the second ORF that encodes the capsid protein precursor, and encodes the 4 amino acids of the N terminal of the capsid protein precursor PPSIV-IRES-NCP (an artificial product manufactured by Genscript), which is a template DNA corresponding to RNA having 12 bases on the 3 ′ side and a gene encoding an internal ribosome binding site (IRES) between them, was added to PrimeSTAR. MAX DNA Polymerase (manufactured by Takara Bio) was used. It was amplified by CR. A firefly luciferase gene (pE01-Luc, hereinafter referred to as “F-Luc”) was also amplified in the same manner. These DNAs were ligated and amplified similarly. Using the obtained DNA, a template DNA having a gene encoding the stem loop structure of FIG. 6 (hereinafter referred to as “5′-SL”) at the 5 ′ end and a T7 promoter sequence for further promoting translation is prepared. did.

  Further, in the same manner as described above, the template DNA does not include the 53 base at the 3 ′ end of the first ORF, that is, the template DNA includes the 6004 to 6204 bases of the PSIV genome; and the gene encoding the IRES. Template DNAs were prepared that were partially defective, ie, containing bases 6017-6204, 6073-6204, and 6101-6204 of the PSIV genome, respectively.

(2) Preparation of template mRNA Using the MEGAscript T7 Kit (manufactured by Applied Biosystems), a template mRNA obtained by transcription of each template DNA was obtained. The obtained template mRNA was purified using RNeasy MinElute Cleanup Kit (manufactured by QIAGEN), and the template mRNA was quantified using absorption of light at a wavelength of 260 nm.

  The obtained 5 types of template mRNA are shown in FIG. The numbers in FIG. 4 indicate base numbers in PSIV genomic RNA. L, SL, and PK indicate the three-dimensional structure of IRES necessary for translation, L indicates a loop, SL indicates a stem-loop, and PK indicates a pseudoknot. 5′-SL has the sequence of SEQ ID NO: 1 (SEQ ID NO: 1), is a stem loop at the 5 ′ end, and has a role of inhibiting translation mediated by the 5 ′ end in eukaryotic cells. . N-CP is 6193-6204 bases in the PSIV genome, 12 bases encoding 4 amino acids at the 5 ′ end of the second ORF encoding the capsid protein precursor and N-terminal of the capsid protein precursor It is. The sequence has a role of promoting IRES-mediated translation. F-Luc is a firefly luciferase gene.

(3) Translation of template mRNA Using the WEPRO1240 Expression Kit (manufactured by CellFree Science), the template mRNA obtained above was cell-free translated. Specifically, a mixture of the above template mRNA (3 pmol), wheat germ extract (WEPRO1240, 2 μL), creatine kinase (final concentration 40 ng / μL) and standard wheat cell-free protein synthesis substrate (final concentration 1 ×) (10 μL) was incubated at 26 ° C. for 1 hour.

(4) Luciferase assay The reaction mixture of the above (3) was diluted 11 times with water, and 5 μL thereof was mixed with Luciferase Assay Reagent containing luciferin (Promega, 75 μL). The reaction mixture was placed in a black 96-well plate, and the fluorescence intensity was measured using a multi-label plate counter (Perkin-Elmer, Wallac ARVO MX). Measurement was performed three times from the above translation, and the average value was calculated. The results are shown in FIG. 5 in terms of relative activity with the value of the second template mRNA (FIG. 4: 2: IRES6004-Luc, hereinafter referred to as “template mRNA2”) being 100%.

  As shown in FIG. 5, the translation efficiency was highest for template mRNA2. Template mRNA2 contains the entire IRES but not part of the first ORF. On the other hand, the translation efficiency is reduced in the template mRNA1 that contains the entire IRES but contains a part of the first ORF. Such a result is considered to be due to a part of the first ORF affecting the three-dimensional structure of the IRES. On the other hand, almost no translational activity was observed in template mRNAs 4 to 5 in which a part of IRES was deleted. From these results, it can be seen that in this system, translation mediated by the 5 'end is almost completely inhibited by 5'-SL.

Experimental Example 2 Inhibition of translation by Anti-IRES In order to control mRNA expression, translation must be suppressed in the absence of a ligand. Therefore, we searched for Anti-IRES that can efficiently inhibit IRES-mediated translation.

  Specifically, by the same method as in Experimental Example 1 (1) to (2), IRES is inserted between the 5 ′ end stem loop (5′-SL) and the IRES of PSIV-IRES shown in FIG. 6 (Xn). MRNA was prepared in which the following sequences complementary to the sequences involved in any of the important three-dimensional structures of loop (L), stem loop (SL), or pseudonodule (PK) were inserted.

  Using the obtained mRNA, translation efficiency was calculated by the same method as in Experimental Example 1 (3) to (4). The results are shown in FIG. 7 by relative activity with the value of template mRNA 2 in Experimental Example 1 set to 100%.

  As shown in FIG. 7, the template mRNA 8 binding to PK-III, the template mRNA 11 binding to cPK-II, and the template mRNA 13 binding to SL-V are very excellent. Particularly, the template mRNA 8 and the template mRNA 13 are important. The inhibitory activity was superior to that of the template mRNA4 lacking the IRES portion. Template mRNA 8 is considered to inhibit IRES-mediated translation by binding to one of the sequences constituting PK-III and changing the three-dimensional structure of IRES.

Experimental Example 3 Examination of Anti-IRES Length Next, the base length of anti-IRES sufficient to inhibit IRES-mediated translation was examined.

  In the same manner as in Experimental Example 1 (1) and (2), a template mRNA was prepared in which the length of the anti-IRES of the template mRNA 8 having the most excellent inhibitory activity in Experimental Example 2 was changed as follows. .

  Using the obtained mRNA, translation efficiency was calculated by the same method as in Experimental Example 1 (3) to (4). The results are shown in FIG. 8 by relative activity with the value of template mRNA 2 in Experimental Example 1 set to 100%.

  As shown in the results of FIG. 8, when the base length of anti-IRES was 4, the inhibitory activity was completely lost. On the other hand, it was revealed that when the base length is 7 to 8, it can be almost completely inhibited.

Experimental Example 4 Restoration of Translation Inhibition by Anti-IRES In order to control the on / off of mRNA expression, it is necessary to restore the IRES-mediated translation by restoring the IRES structure changed by the ligand. Then, anti-anti-IRES which couple | bonds with anti-IRES complementarily and inhibits the function was examined.

  By the same method as in Experimental Example 1 (1) to (2), in the template mRNA 8 of Experimental Example 2, between 5′-SL and anti-IRES (Yn), via 5 bases (loop) A template mRNA having the following anti-anti-IRES having different base lengths was prepared.

  Using the obtained mRNA, translation efficiency was calculated by the same method as in Experimental Example 1 (3) to (4). The results are shown in FIG. 9 in terms of relative activity with the value of template mRNA 2 in Experimental Example 1 set to 100%.

  As shown in the results of FIG. 9, when the complementary base length was 3 to 5, IRES-mediated translation inhibited by anti-IRES could not be recovered. However, when the complementary base length is 7, the IRES-mediated translation can be restored to almost 50%. When the anti-IRES having the same base length as the anti-IRES is used, the IRES is reduced to about 80%. Intervening translation could be restored. Thus, it was clarified that if an anti-IRES and an anti-anti-IRES form a double strand, the IRES structure can be restored and IRES-mediated translation can be restored.

Example 1
Based on the results of Experimental Examples 1 to 4, the following strategy was devised as a system for controlling on / off of mRNA expression by a ligand.
-5'-SL is provided at the 5 'end to suppress 5'-end-mediated translation and use IRES-mediated translation.-Aptamer sequences that can bind strongly and selectively to ligands include anti-IRES and anti-anti -Insertion between the IRES-Modulator sequence (MS) is inserted 5 'to the anti-anti-IRES to control the formation of anti-IRES / anti-anti-IRES duplexes-Translation efficiency An example based on the above strategy of optimizing the modulator arrangement to increase the on-off ratio is shown in FIG.

  The left figure of FIG. 10 shows the case where no ligand is present, and the modulator sequence (MS) binds to the 3 ′ end of the anti-anti-IRES and the 5 ′ end of the aptamer sequence to form a stem loop (MS-SL). is doing. As a result, since anti-anti-IRES cannot bind to anti-IRES, anti-IRES binds to an important site of IRES and changes the three-dimensional structure of IRES. Therefore, 5 'end-mediated translation is inhibited by 5'-SL and IRES-mediated translation is also suppressed, so mRNA is not translated.

  On the other hand, when there is a ligand that binds tightly and selectively to the aptamer (right diagram in FIG. 10), the modulator sequence (MS) is released from the aptamer and anti-anti-IRES by binding of the ligand to the aptamer, and anti- Anti-IRES and anti-IRES can form a duplex, and as a result, the three-dimensional structure of IRES is restored and IRES-mediated translation proceeds.

  In order to realize the above strategy, an mRNA capable of using theophylline as a ligand is designed based on the template mRNA aa8 of Experimental Example 4 and the same method as in Experimental Examples 1 (1) and (2) above. Synthesized. Specifically, in template mRNA aa8, a theophylline aptamer sequence is inserted between anti-anti-IRES and anti-IRES, and a modulator sequence (MS) is inserted between anti-anti-IRES and 5′-SL. ) Was inserted.

  At this time, in the modulator sequence (MS), the portion that binds to anti-anti-IRES was fixed to 4 bases of 5'-AGAC-3 ', and the portion that bound to the aptamer sequence was examined. The reason for fixing the portion that binds to anti-anti-IRES to 4 bases is that the 3 ′ side of the modulator sequence (MS) must be the same as the 5 ′ side of the anti-IRES, so the modulator sequence There is a possibility that (MS) itself binds to IRES and the translation is inhibited, but as shown in template mRNAs 8-4 and 8-r4 in Experimental Example 3 above, if the length is 4 bases, the modulator sequence (MS) is IRES. This is because it does not unconditionally inhibit translation.

  Moreover, the part (Zn) couple | bonded with an aptamer sequence among modulator arrangement | sequences (MS) designed 2-8 base length as follows, and the modulator sequence (MS) whole designed 6-12 base length.

  Using the above mRNA, the translation efficiency was calculated by the same method as in Experimental Example 1 (3) to (4) in the presence and absence of 1 mM theophylline. The results are shown in FIG. 11 by relative activity with the value of template mRNA 2 in Experimental Example 1 set to 100%.

  As shown in the results of FIG. 11, if the portion of the modulator sequence (MS) that binds to the aptamer sequence is 5 bases or more in length and 9 bases or more in total, translation in the absence of theophylline as a ligand is sufficiently suppressed. It had been. This is probably because the modulator sequence (MS) is bound to the anti-anti-IRES and the aptamer sequence, so that the anti-IRES is bound to the IRES and changes its three-dimensional structure. On the other hand, when the portion of the modulator sequence (MS) that binds to the aptamer sequence is 8 bases long and 12 bases in total, translation did not proceed sufficiently even if theorifin was present. The reason is considered to be that the modulator sequence (MS) is not separated from the anti-anti-IRES and the aptamer sequence even when theorifin is present, and the anti-IRES is not separated from the IRES.

  On the other hand, when the portion of the modulator sequence (MS) that binds to the aptamer sequence is 5 to 7 bases long, and the total length is 9 to 11 bases, the ratio of the presence of theophylline to the absence of theophylline ( (ON / OFF ratio) is very high from about 8 to 10 times. Thus, according to the present invention, it was proved that gene expression can be controlled by a ligand. In addition, although a cell-free eukaryotic gene expression system was used in this example, the same result can be obtained even by a transformation technique using the eukaryotic cell itself because good results were obtained in this mode. it is conceivable that.

  In each mRNA, the Gibbs free energy (ΔG) of the modulator sequence stem loop (MS-SL), which is most important for on / off control of mRNA expression, was calculated from the RNA structure. The results are shown in FIG.

  As shown in FIG. 11, if the Gibbs free energy (ΔG) is too large, the modulator array stem loop is likely to be unstable, and IRES-dependent translation is initiated even in the absence of a ligand, whereas if it is too small, the modulator array It has been found that the stem loop is over-stabilized and IRES-dependent translation may not be initiated in the presence of ligand.

Example 2
Based on the results of Example 1 above, mRNA was designed to apply a system using theophylline as a ligand to combinations of other ligands and aptamer sequences.

  Specifically, in the mRNA using the combination of flavin mononucleotide (FMN) -aptamer, tetracycline (tc) -aptamer and sulforhodamine B-aptamer in addition to the theorifin-aptamer of FIG. 12, the modulator sequence stem loop (MS- The modulator sequence (MS) is designed so that the Gibbs free energy (ΔG) of SL) is close to −11.7 kcal / mol of theo5, which is the mRNA having the highest ON / OFF ratio in Example 1 above. Prepared in the same manner as in the methods of Examples 1 (1) and (2) above. However, if the concentration of tetracycline or the like is too high, IRES-mediated translation may be inhibited. Therefore, only the theophylline concentration was 1 mM, and the other concentrations were 0.3 mM. The modulator sequence (MS) and Gibbs free energy (ΔG) in each mRNA are shown below.

  Using the mRNA, the translation efficiency was calculated by the same method as in Experimental Examples 1 (3) to (4) in the presence and absence of each ligand. The results are shown in FIG. 13 by relative activity with the value of template mRNA 2 in Experimental Example 1 set to 100%.

  As shown in the results of FIG. 13, the translation efficiency when each ligand was not used was as low as that of mRNA theo5, while the translation efficiency when each ligand was used was improved. In particular, when tetracycline was used as a ligand, the ON / OFF ratio was extremely high, reaching about 30.

Claims (11)

An RNA molecule capable of controlling expression of a target gene by a ligand, wherein the RNA molecule has the following structure and sequence from the 5 'side.
(A) a 5 ′ end stem loop that inhibits 5 ′ end dependent translation;
(B) a modulator sequence that forms a modulator sequence stem loop by complementary binding to a portion of the aptamer sequence described below in the absence of a ligand;
(C) An anti-anti-internal ribosome that forms a stem loop together with a modulator sequence and a part of the following aptamer sequence in the absence of a ligand and forms a complementary duplex with the anti-internal ribosome binding site sequence in the presence of the ligand Binding site sequence;
(D) an aptamer sequence capable of binding to a ligand;
(E) In the absence of a ligand, a complementary duplex is formed with a part of the internal ribosome binding site to inhibit the binding of the ribosome to the internal ribosome binding site, and in the presence of the ligand, an anti-anti-internal ribosome binding site sequence An anti-internal ribosome binding site sequence forming a complementary duplex;
(F) an internal ribosome binding site; and (g) a gene of interest.   The RNA molecule according to claim 1, wherein the anti-internal ribosome binding site sequence has a complementary base length of 6 or more and 12 or less with respect to the internal ribosome binding site.   The RNA molecule according to claim 1 or 2, wherein the complementary base length of the anti-anti-internal ribosome binding site sequence with respect to the anti-internal ribosome binding site sequence is 6 or more and 12 or less.   The RNA molecule according to any one of claims 1 to 3, wherein the complementary base length of the modulator sequence for a part of the anti-anti-internal ribosome binding site sequence is 5 or less.   The RNA molecule according to any one of claims 1 to 4, wherein a complementary base length of the modulator sequence with respect to a part of the aptamer sequence is 4 or more and 8 or less.   The RNA molecule according to any one of claims 1 to 5, wherein a Gibbs free energy (ΔG) of the modulator sequence stem-loop structure is -17.5 kcal / mol or more and -8.5 kcal / mol or less.   The RNA molecule according to any one of claims 1 to 6, wherein the internal ribosome-binding site is that of the Chabaine stink bug enterovirus.   The RNA molecule according to claim 7, wherein the anti-internal ribosome binding site sequence is capable of binding to pseudo-nodule III or stem loop V in the three-dimensional structure of the internal ribosome binding site sequence of Chabaenka Stink bug enteric virus.   A DNA molecule encoding the RNA molecule according to any one of claims 1 to 8, and having a promoter sequence upstream of the coding region. A method for controlling the expression of a target gene in a eukaryotic cell, comprising:
A method comprising mixing and reacting the RNA molecule according to any one of claims 1 to 8 or the DNA molecule according to claim 9 with an eukaryotic cell extract. A method for controlling the expression of a target gene in a eukaryotic cell, comprising:
A method comprising transfecting a eukaryotic cell with the DNA molecule of claim 9 and activating an internal ribosome binding site by introducing a ligand into the eukaryotic cell.