JP6877752B2 - Method for identifying intracellular proteins - Google Patents

Description

The present invention relates to a method for identifying an endogenous protein in a cell, a stabilized aptamer that can be used therefor, and a method for identifying a cell differentiation and reprogramming state.

An artificial gene expression control system that functions only in target cells can be a useful tool in biotechnology and medical applications. The cell type and state are mainly determined by proteins. Therefore, it is important to develop a mechanism that can detect proteins in human cells and control gene expression.

An mRNA switch is a gene switch that has an RNA (aptamer) region that binds to a specific protein in the 5'UTR of mRNA. This switch suppresses its own translation by binding proteins to aptamers on mRNA. In principle, by incorporating an aptamer that binds to an arbitrary protein, an mRNA switch that detects the desired protein and suppresses translation can be produced. In addition, direct introduction of mRNA into cells has higher transfer efficiency and lower risk of genome damage than conventional gene transfer using a plasmid. Therefore, the mRNA switch is ideal as an artificial gene expression control system that functions only in target cells.

The following two examples are known as gene switches that target proteins expressed in human cells, which are produced by incorporating aptamers into mRNA. One is a switch in which a part of the ribozyme incorporated into the 3'UTR is converted into an aptamer of the human U1A protein (see Non-Patent Document 1). However, in this study, U1A was overexpressed by plasmid introduction, and no response to proteins endogenous to human cells was realized. The other is a switch that controls splicing in the presence of a target protein and produces different mature mRNAs by incorporating an aptamer into the intron (see Non-Patent Document 2). This switch can control the expression of fluorescent proteins and prodrug activators in response to NF-kB and CTNNB1 (β-catenin). However, in this study, the target protein is limited to the nuclear localization protein because the protein that binds to the aptamer must be present in the nucleus. In addition, since splicing control is used, it is difficult to introduce a system other than DNA such as a plasmid or a viral vector.

From the above, it is desired to develop a switch capable of introducing mRNA without limiting the target protein and detecting the expression of the endogenous protein. Since mRNA controls gene expression by translation control, it is possible to realize switch function control by direct introduction of mRNA, and mRNA that detects microRNA and controls translation has already been developed (Non-Patent Document 3). However, the development of an mRNA switch capable of detecting human endogenous proteins and controlling translation has not yet been realized.

Auslander, S. et al., Nat. Methods 11, 1154-60 (2014) Culler, S. J. et al., Science. 330, 1251-5 (2010). Wroblewska, L. et al., Nat. Biotechnol. 1-5 (2015)

The mRNA switches produced so far have not been able to detect human endogenous proteins. There is a need for a method for detecting human cell endogenous proteins in living cells without destroying the cells.

The present inventors have discovered that human cell endogenous proteins can be specifically and quantitatively detected in living cells by introducing mRNA having an aptamer sequence specific to the intracellular protein into cells. Furthermore, the present inventors design an mRNA having an aptamer sequence for an endogenous protein specific to an induced pluripotent stem (iPS) cell and an aptamer sequence obtained by stabilizing the aptamer sequence, and introduce the mRNA into the cell. Therefore, it was discovered that the differentiation state and the reprogramming state of cells can be detected, and the present invention has been completed.

Therefore, the problem of the present invention can be solved by the following means.
[1] A method for identifying an endogenous protein in a cell, which comprises the following steps:
(1) A step of introducing an intracellular protein-responsive mRNA containing an aptamer sequence specific to an intracellular protein and a marker gene sequence functionally linked thereto or a vector encoding the mRNA into a desired cell; and ( 2) A step of identifying an intracellular protein based on the amount of translation of the marker gene.
[2] A method for identifying cells, which includes the following steps:
(1) A step of introducing an intracellular protein-responsive mRNA containing an aptamer sequence specific to an intracellular protein and a marker gene sequence functionally linked to the aptamer sequence or a vector encoding the mRNA into a desired cell;
(2) A step of identifying cells expressing the protein based on the amount of translation of the marker gene.
[3] Based on the RNA secondary structure formed by the natural LIN28A aptamer sequence represented by SEQ ID NO: 1, the structure-stabilized LIN28A aptamer obtained by adding one or more of the following modifications:
(1) Replace the AU base pairs constituting the stem containing the 5'-terminal base and the 3'-terminal base with GC base pairs;
(2) Replace the AU base pairs that make up the stem located between the two loops with GC base pairs; or (3) Add 1 to 5 base pairs to the stem located between the two loops.
[4] A structurally stabilized LIN28A aptamer represented by any of SEQ ID NOs: 2 to 4.
[5] Based on the RNA secondary structure formed by the natural U1A aptamer sequence represented by SEQ ID NO: 6, the structure-stabilized U1A aptamer obtained by making the following modifications:
(1) Add 1 to 15 AU base pairs or GC base pairs to a stem structure containing 5'end bases and 3'end bases, or add 1 to 15 AU base pairs to a stem existing between two boxes. Alternatively, a GC base pair is added; or a structure-stabilized U1A aptamer is obtained by making the following modifications based on the RNA secondary structure formed by the U1A aptamer sequence represented by SEQ ID NO: 8.
(2) Add 1 to 15 AU base pairs or GC base pairs to a stem containing 5'-terminal bases and 3'-terminal bases.
[6] Based on the RNA secondary structure formed by the p50A aptamer sequence represented by SEQ ID NO: 83, the following modifications are made to stabilize the p50A aptamer:
Add 1 to 20 AU base pairs or GC base pairs to the stem structure containing 5'end bases and 3'end bases.
[7] A structurally stabilized U1A aptamer represented by any of SEQ ID NOs: 7, 9, 10 and 84.
[8] An mRNA containing the structurally stabilized aptamer sequence according to any one of [3] to [7] and a marker gene sequence functionally linked thereto.
[9] A method for identifying the differentiation state of cells, which includes the following steps:
(1) Introduce an mRNA containing an aptamer sequence specific to a protein expressed by an undifferentiated cell and a marker gene sequence functionally linked to the aptamer sequence, or a vector encoding the mRNA into a cell group that can contain pluripotent stem cells. Steps; and (2) A step of identifying the differentiation state of cells based on the amount of translation of the marker gene.
[10] The method according to [9], wherein the aptamer sequence is a natural LIN28A aptamer sequence or a structurally stabilized aptamer sequence according to [3] or [4].

According to the present invention, it is possible to detect endogenous proteins of living cells alive with high sensitivity and quantitatively, which was impossible until now. Also, to identify induced pluripotent stem (iPS) cells by detecting endogenous proteins in living cells, and to detect their differentiation and reprogramming states, and to classify, isolate or selectively eliminate them. Is now possible. Furthermore, we were able to obtain stabilized aptamers that are particularly useful for these detections. These stabilized aptamers can be used as protein-specific sequences provided in the mRNA used in the above-mentioned method, and also optionally bind to other molecules to strongly bind to the target protein in the cell and their functions. Can be useful as an anti-cancer agent or antagonist, or as a sensor extracellularly.

FIG. 1 is a diagram illustrating the mechanism of action of a protein-responsive mRNA switch. The protein-responsive switch exists in the 5'untranslated region (5'-UTR) of mRNA as a sequence (aftermer) that binds to a specific protein, and the target protein can be obtained by binding to the trigger protein. It suppresses the translation of. That is, in the presence of the trigger protein, the translation of the mRNA switch is suppressed (Fig. 1, upper right), and in the absence, normal translation is performed (Fig. 1, lower right). This molecular device is artificially made by inserting an aftermer into the 5'-UTR of mRNA. FIG. 2 is a diagram showing an outline of the plasmid used in the experiment. Map information of the plasmids used as empty vectors for the switch plasmid (top) and trigger plasmid (bottom) was depicted using ApE (http://www.biology.utah.edu/jorgensen/wayned/ape/). All plasmids are transcribed from the CMV promoter. The switch plasmid was inserted with an aptamer sequence at a restriction enzyme site 14 bases downstream from the transcription initiation site. The transcript of the trigger plasmid is bicistronically translated with tagRFP along with the translation of the trigger protein. The trigger protein and tagRFP are translated as two independent proteins by ribosome skipping occurring at the T2A site. Therefore, the expression level of the trigger protein and the expression level of tagRFP are considered to be the same. FIG. 3 is a secondary structure prediction diagram of the LIN28A aptamer used in the experiment. FIG. 4 is a graph showing that each switch plasmid responds to LIN28A in HEK293 FT cells and is translated-suppressed. FIG. 5 is a graph showing that the switch plasmid in which stbC is inserted responds to LIN28A even in HeLa cells and translation is suppressed. FIG. 6 is a graph showing that translation is suppressed by the specific binding of the switch plasmid in which stbC is inserted to LIN28A. FIG. 7 is a series of diagrams showing that the translation efficiency of the prepared switch changes according to the amount of LIN28A in the cell. FIG. 7A is a graph showing that the translation efficiency of the switch decreases depending on the amount of Lin28A expressed in a concentration-dependent manner of doxycycline. FIG. 7B shows the results of confirming the expression levels of Lin28A and GAPDH at each doxycycline concentration by Western blotting. FIG. 7C is a graph showing the relationship between the relative expression level of LIN28A and the doxycycline concentration by quantifying the relative expression level of LIN28A from the band concentration of FIG. 7B. FIG. 7D is a graph showing the relationship between the relative expression level of LIN28A and the fluorescence ratio of each switch. FIG. 8 is a series of diagrams showing that the prepared LIN28A switch also responds to LIN28A by mRNA transfection. FIG. 8A is a dot plot showing the cell population at each LIN28A mRNA introduction level for the stbC-inserted LIN28A switch. FIG. 8B is a dot plot showing that in a LIN28A switch with stbC inserted, populations are segregated according to the amount of LIN28A mRNA introduced. The stbC dot plot is a superposition of the dot plots shown in Figure 8A. Figure 8C is a graph showing that the LIN28A switch with stbC inserted is translated-suppressed depending on the amount of LIN28A. FIG. 9 is a series of diagrams illustrating a mechanism for separating iPS cells and differentiated cells using a LIN28A switch. FIG. 9A is a diagram schematically explaining that the translation of the LIN28A switch is suppressed in iPS cells. FIG. 9B is a diagram illustrating that cells into which a LIN28A switch has been introduced can be divided into iPS cells and differentiated cells by flow cytometry. FIG. 10 is a series of diagrams showing that the LIN28A switch with stbC inserted undergoes translational repression in iPS cells. FIG. 10A is a diagram showing that iPS cells and differentiated cells can be distinguished on a dot plot plane of flow cytometry using a LIN28A switch. FIG. 10B is a histogram showing that the stbC-inserted LIN28A switch undergoes translational repression in iPS cells rather than in differentiated cells. FIG. 10C is a graph showing how much the mRNA into which each aptamer was inserted suppressed translation in iPS cells as compared with differentiated cells. FIG. 11 is a series of diagrams showing that the translation efficiency of the LIN28A switch changes with the progress of differentiation induction. FIG. 11A is a graph comparing the translation efficiencies of iPS cells and cells of each differentiation induction day. FIG. 11B is a dot plot showing the results of flow cytometry of each cell and its superposition. FIG. 12 is a secondary structure prediction diagram of the U1A aptamer used in the experiment. FIG. 13 is a graph showing that each switch plasmid responds to U1A in HEK293 FT cells and is translated-suppressed. FIG. 14 is a series of diagrams showing that the prepared U1A switch responds to U1A endogenous to human cells and undergoes translational repression. FIG. 14A is a fluorescence photograph of the U1A switch and its mutant introduced into cells. FIG. 14B is a histogram showing the fluorescence amount of the U1A switch and its mutant. FIG. 14C is a graph showing the fluorescence ratio of each U1A switch. FIG. 15 is a graph showing the relative translation efficiency at the time of introducing shRNA in the U1A switch. FIG. 16 is a graph showing the expression level of endogenous miRNA at the time of introduction of the LIN28A switch. FIG. 17 is a secondary structure prediction diagram of the p50 aptamer used in the experiment. FIG. 18 is a graph showing that each switch plasmid responds to p50 in HEK293 FT cells and is translated-suppressed.

Hereinafter, the present invention will be described in detail with reference to embodiments. The following embodiments do not limit the present invention.

[First Embodiment: Method for detecting intracellular protein]
The present invention relates to a method for identifying intracellular proteins, according to the first embodiment. The detection method includes the following steps.
(1) A step of introducing an intracellular protein-responsive mRNA containing an aptamer sequence specific to an intracellular protein and a marker gene sequence functionally linked thereto or a vector encoding the mRNA into a desired cell; and ( 2) A step of identifying an intracellular protein based on the amount of translation of the marker gene.

Here, the desired cell is not particularly limited, and the cell may be arbitrary. For example, it may be a cell included in a cell group collected from a multicellular organism, or may be a cell included in a cell group obtained by culturing an isolated cell. The cells are particularly cells collected from mammals (eg, humans, mice, monkeys, pigs, rats, etc.), cells isolated from mammals, or cells obtained by culturing a mammalian cell line. It may be there. Examples of somatic cells include keratinizing epithelial cells (eg, keratinized epidermal cells), mucosal epithelial cells (eg, epithelial cells on the surface of the tongue), exocrine gland epithelial cells (eg, mammary cells), hormone-secreting cells (eg, mammary cells). , Adrenal medulla cells), cells for metabolism and storage (eg, hepatocytes), luminal epithelial cells that make up the interface (eg, type I alveolar cells), luminal epithelial cells of the inner chain canal (eg, blood vessels) Endophilic cells), cell with ciliary capacity (eg, airway epithelial cells), cells for extracellular matrix secretion (eg, fibroblasts), contractile cells (eg, smooth muscle cells), blood and immune system Cells (eg, T lymphocytes), sensory cells (eg, rod cells), autonomic nervous system neurons (eg, cholinergic neurons), sensory and peripheral neuronal support cells (eg, companion cells), central nervous system Nerve cells and glial cells (eg, stellate glial cells), pigment cells (eg, retinal pigment epithelial cells), and their precursor cells (tissue precursor cells). There are no particular restrictions on the degree of cell differentiation or the age of the animal from which the cells are collected, and the same applies to undifferentiated progenitor cells (including somatic stem cells) and final differentiated mature cells. It can be used as the origin of somatic cells in the invention. Examples of undifferentiated progenitor cells include tissue stem cells (somatic stem cells) such as neural stem cells, hematopoietic stem cells, mesenchymal stem cells, and dental pulp stem cells. In the present invention, the individual mammal from which the somatic cells are collected is not particularly limited, but is preferably human. Further, the cell may be a cell that has been artificially manipulated after collecting the early somatic cells.

Further, in the present invention, "identifying an endogenous protein in a cell" means detecting, identifying, classifying, and selecting an endogenous protein in a cell by using the cell alive. Alternatively, the present invention can be said to be a method for identifying cells, and in this case, a method for identifying a target cell using a specific cell endogenous protein as an index. Then, for cell identification, after identification, the cell type expressing the protein is identified, classified, isolated, removed, alive or dead is determined, and a specific biological signal of the cell type is detected. Alternatively, it may include quantification and fractionation based on the specific physical or chemical signal of the cell type.

In step (1) of this embodiment, intracellular protein-responsive mRNA is used. In the present specification, intracellular protein-responsive mRNA may be referred to as protein-responsive mRNA or mRNA switch. Protein-responsive mRNAs include (i) an aptamer sequence specific for an endogenous protein in the cell and (ii) a marker gene sequence functionally linked to it.

(i) Cell-endogenous protein-specific aptamer sequence The cell-endogenous protein-specific aptamer sequence may be any RNA aptamer sequence that specifically binds to the endogenous protein in the desired cell. Therefore, it may be a natural aptamer sequence already known in the literature or the like, an artificial aptamer sequence modified thereto, or another artificially prepared aptamer sequence. The natural aptamer sequence can be appropriately selected from, for example, the database available on the website: (Apta-Index ™: http://www.aptagen.com/aptamer-index/aptamer-list.aspx). The artificial aptamer sequence can be obtained by a method of selecting RNA that binds to a protein of interest from an RNA population containing a random sequence (reference [3]). This method is known as an evolutionary engineering method known as the in vitro selection method or the SELEX method. Therefore, an aptamer that binds to the desired protein can be obtained from the artificial RNA sequence. Then, by incorporating these aptamers into mRNA, an mRNA switch that responds to a desired protein can be created. Specific aptamers and stabilized aptamers suitable for the method of the present invention will be described in detail in the second and third embodiments.

(ii) Marker gene sequence functionally linked to it The "marker gene" in (ii) used in the present invention is any arbitrary gene that is translated intracellularly and functions as a marker to enable identification of the cell. A gene that encodes a protein. Examples of proteins that can be translated intracellularly and function as markers include fluorescence, luminescence, coloration, or visualization and quantification by assisting fluorescence, luminescence, or coloration of fluorescence, fluorescent proteins, and the like. It includes, but is not limited to, a protein encoding a protein that can be transformed, a membrane protein, or a gene encoding a protein that kills a cell by expression, such as an apoptosis-inducing gene or a suicide gene. An apoptosis suppressor gene can also be used as a marker gene in combination with an apoptosis-inducing gene. In the present specification, a protein translated from mRNA containing a nucleic acid corresponding to the coding region of the marker gene is referred to as a marker protein.

In the present invention, the fluorescent proteins include blue fluorescent proteins such as Sirius, BFP and EBFP; cyan fluorescent proteins such as mTurquoise, TagCFP, AmCyan, mTFP1, MidoriishiCyan and CFP; TurboGFP, AcGFP, TagGFP and Azami-Green (for example, hmAG1). ), ZsGreen, EmGFP, EGFP, GFP2, HyPer and other green fluorescent proteins; TagYFP, EYFP, Venus, YFP, PhiYFP, PhiYFP-m, TurboYFP, ZsYellow, mBanana and other yellow fluorescent proteins; KusabiraOrange (eg hmKO2), mOrange Orange fluorescent protein such as TurboRFP, DsRed-Express, DsRed2, TagRFP, DsRed-Monomer, AsRed2, mStrawberry; Red fluorescent protein such as TurboFP602, mRFP1, JRed, KillerRed, mCherry, HcRed, KeimaRed (eg hdKeimaRed), mrAsberry Examples include, but are not limited to, near-infrared fluorescent proteins such as mPlum.

In the present invention, the photoprotein can be exemplified by aequorin, but is not limited thereto. In addition, examples of proteins that assist fluorescence, luminescence, or color development include, but are not limited to, enzymes that decompose fluorescence, luminescence, or color development precursors such as luciferase, phosphatase, peroxidase, and β-lactamase. Here, in the present invention, when a substance that assists fluorescence, luminescence, or color development is used as a marker gene, in selecting cells in which a specific protein is present or not, the corresponding precursor is brought into contact with the cell. Alternatively, it can be done by introducing the corresponding precursor into the cell.

In the present invention, the apoptosis-inducing gene means a gene encoding a protein having an apoptosis-inducing activity on cells. For example, IκB, Smac / DIABLO, ICE, HtrA2 / OMI, AIF, endonuclease G, Bax, Bak, Noxa, Hrk (harakiri), Mtd, Bim, Bad, Bid, PUMA, activated caspase-3, Fas, Tk, etc. These include, but are not limited to. In the present invention, Bim is preferably used as an apoptosis-inducing gene.

In the present invention, the suicide gene means a gene whose expression in a cell is lethal to the cell. In the present invention, the suicide gene may be one that causes cell death by itself (eg, diphtheria A toxin), or one whose expression of this gene makes the cell sensitive to a particular drug (eg,). , The expression of the simple herpestimidine kinase gene makes the cell sensitive to antiviral compounds). Examples of suicide genes include diphtheria A toxin, simple herpesthymidine kinase gene (HSV-TK), carboxypeptidase G2 (CPG2), carboxylesterase (CA), cytosine deaminase (CD), cytochrome P450 (cyt-450), and deoxycytidine. Genes encoding kinase (dCK), nitroreductase (NR), purine nucleoside phosphorylase (PNP), thymidine phosphorylase (TP), thymidine varicella virus thymidine kinase (VZV-TK), xanthin-guanine phosphoribosyl transferase (XGPRT), etc. However, it is not limited to these. In the present invention, HSV-TK is preferably used as the suicide gene.

In the present invention, the marker gene may include a gene encoding a localization signal. Examples of the localization signal include a nuclear localization signal, a cell membrane localization signal, a mitochondrial localization signal, a protein secretion signal, and the like, and specifically, a classical nuclear localization sequence (NLS), M9. Sequences, mitochondrial target sequences (MTS), endoplasmic reticulum translocation sequences, but are not limited to these. Such a localization signal is used when a step of detecting cells in which a specific protein is present or absent by imaging cytometry or the like, which will be described later, and further separating / selecting such cells is performed on an image. Especially advantageous.

In the present invention, the functional linkage between the aptamer sequence and the marker gene sequence is defined as within the 5'UTR, 3'UTR, and / or of the open reading frame (including the start codon) encoding the marker gene. It means that the aptamer sequence is provided in the open reading frame. The protein-responsive mRNA preferably comprises a Cap structure (7 methylguanosine 5'phosphate), an open reading frame encoding a marker gene, and a poly A tail in the 5'to 5'to 3'direction from the 5'end. , 5'UTR, 3'UTR, and / or an aptamer sequence in an open reading frame. The position of the aptamer sequence in the mRNA may be 5'UTR, 3'UTR, or within the open reading frame (3'side of the start codon).

Preferably, the protein-responsive mRNA has the nucleic acid sequences of (i) and (ii) linked in this order in the 5'to 3'direction. At this time, the number of bases and the type of bases between the cap structure and the aptamer sequence may be arbitrary as long as they do not constitute a stem structure or a three-dimensional structure. For example, the number of bases between the cap structure and the aptamer sequence can be designed to be 0 to 50 bases, preferably 0 to 20 bases. The number of bases between the aptamer sequence and the start codon and the type of base may be arbitrary as long as they do not constitute a stem structure or a three-dimensional structure, and the number of bases between the aptamer sequence and the start codon is 0 to 50. It can be designed with a base, preferably 10 to 30 bases.

In the present invention, it is preferable that AUG serving as a start codon does not exist in the aptamer sequence. For example, if the aptamer sequence is present in the 5'UTR and the aptamer sequence contains AUG, it should be designed to be in-frame in relation to the marker gene linked to the 3'side. Is preferable. Alternatively, if the aptamer sequence contains the AUG, the AUG can be converted to another triplet sequence if it is not important for binding to the target. If AUG is present in the stem structure and replaces the base, it also replaces the corresponding base so that it forms a base pair after the replacement. In addition, in order to minimize the influence of AUG in the aptamer sequence, the location of the aptamer sequence in the 5'UTR can be changed as appropriate. For example, the number of bases between the cap structure and the AUG sequence in the aptamer sequence is 0 to 60 bases, for example, 0 to 15 bases, 10 to 20 bases, 20 to 30 bases, 30 to 40 bases, 40 to 50 bases. , 50-60 bases can be designed. However, it is desirable that the aptamer insertion position is close to the cap structure.

Such mRNA can be genetically engineered once its nucleic acid sequence is designed. For example, it can be prepared by the PCR method based on a predetermined primer and template DNA, but is not limited to a specific preparation method. Further, with respect to a vector encoding such mRNA, if a predetermined mRNA sequence is designed and determined, it can be appropriately prepared using a commercially available DNA vector system.

Next, each step of the present invention will be described. The step (1) is a step of introducing a protein-responsive mRNA or a vector encoding the mRNA into a desired cell (hereinafter, may be referred to as an introduction step).

The step of introducing the protein-responsive mRNA can be carried out by using a lipofection method, a liposome method, an electroporation method, a calcium phosphate co-precipitation method, a DEAE dextran method, a microinjection method, a gene gun method, or the like. Optionally, two, three, or four or more protein-responsive mRNAs having different aptamer sequences and marker gene sequences can be co-introduced. Further, at this time, in some cases, the control mRNA can be co-introduced into the target cell. The control mRNA is an mRNA that does not have an aptamer sequence and encodes a marker gene different from the marker gene encoded by the protein-responsive mRNA. The activity ratio of marker proteins expressed from two or more co-introduced mRNAs is constant within the cell population. Further, the amount to be introduced at this time varies depending on the cell group to be introduced, the mRNA to be introduced, the method of introduction and the type of the reagent to be introduced, and those skilled in the art can appropriately select these in order to obtain a desired translation amount. The amount of control mRNA introduced can also be appropriately selected by those skilled in the art in order to obtain the desired translation amount.

The introduction of protein-responsive mRNA can also be carried out by introducing a vector encoding the mRNA. Introduction with a more stable DNA vector is particularly useful when the introduced cells are not subsequently used for biological transplantation or the like, for example, for screening or testing purposes. The introduction of the vector into cells can be carried out by the same means as the introduction of mRNA. The same method can be used when introducing a vector encoding two or more protein-responsive mRNAs having different aptamer sequences and marker gene sequences, or when introducing a vector encoding a control mRNA.

When a protein-responsive mRNA is introduced into a cell, if an aptamer-specific protein is present in the cell, the protein binds to the aptamer to control the amount of translation of the marker gene encoded by the protein-responsive mRNA. For example, the amount of translation is suppressed. Then, the amount of translation is controlled quantitatively according to the abundance of protein in the cell. On the other hand, when the protein is absent in the cell, the translation amount of the marker gene encoded by the protein-responsive mRNA is not suppressed. Therefore, the amount of translation of the marker gene differs between the cell in which the protein is present or expressed and the cell in which the protein is present or not expressed. Control mRNA, on the other hand, expresses a marker protein regardless of the presence of the protein. This is because even if the control mRNA is introduced into the cell, the control mRNA does not have an aptamer sequence, so that the translation is not regulated according to the presence and / or amount of a predetermined protein.

The subsequent step (2) is a step of identifying the intracellular protein based on the translation amount of the marker gene (hereinafter, may be referred to as an identification step). In the identification step, the intracellular protein is identified based on the translation amount of the marker gene as described above. That is, when an intracellular protein specific to the aptamer sequence contained in the protein-responsive mRNA is present in the cell, the intracellular protein is identified based on the phenomenon that the translation amount of the marker gene is suppressed. In the "identifying" aspect, the presence or absence of suppression of translation of the marker gene is detected, the presence or absence of a predetermined intracellular protein is determined, and the translation suppression efficiency of the marker gene is measured to quantify the intracellular protein. Further, when the marker gene is a drug resistance gene or the like, the presence of the intracellular protein can be confirmed based on the life or death of the cell after culturing in the presence of the drug. Is not limited. Other aspects of discrimination include control of target cell fate by a marker gene that induces apoptosis, cell differentiation by a differentiation-inducing gene or a reprogramming-inducing gene, and detection of reprogramming cells.

Specifically, the identification step can be carried out by detecting a signal from the marker protein using a predetermined detection device. Examples of the detection device include, but are not limited to, a flow cytometer, an imaging cytometer, a fluorescence microscope, a light emitting microscope, a CCD camera, and the like. As such a detection device, those suitable for those skilled in the art can be used depending on the marker protein and the mode of detection. For example, when the marker protein is a fluorescent protein or a luminescent protein, the marker protein can be quantified using a detection device such as a flow cytometer, an imaging cytometer, a fluorescent microscope, or a CCD camera. In the case of a protein that assists luminescence or color development, a method for quantifying the marker protein using a detection device such as a luminescence microscope, a CCD camera, or a luminometer is possible. A cell surface protein-specific detection reagent such as an antibody and a method for quantifying a marker protein using the above-mentioned detection device are possible. In addition, an identification method such as a magnetic cell separator (MACS) that does not undergo a quantification process of a marker protein, and an identification method that substantially involves isolating cells is also possible, and the marker protein is a drug resistance gene. In some cases, a method of identification is possible by isolating live cells based on the expression of the marker gene by drug administration.

Flow cytometry is an example of a preferred identification (detection) method when the marker protein is a fluorescent protein. Flow cytometry can provide the intensity of light emitted by fluorescent proteins and luciferases, which are marker proteins translated in individual cells, as identification information, and in particular, the fluorescence intensity ratio measured by a flow cytometer. Identification information can be obtained by the measurement of. For example, when a fluorescent protein is used, cells having different translation amounts of marker genes are observed as separate bands on a dot plot of flow cytometry. That is, since cells are separated based on the amount of endogenous protein detected on the dot plot of flow cytometry, it is also possible to physically separate these cells after detection, which is also one of the identifications. It may be an embodiment. Alternatively, it is also effective to obtain identification information by measuring the fluorescence intensity ratio by cell image analysis using an imaging cytometer or the like.

The method for measuring the translation amount of protein-responsive mRNA is not limited to the above-mentioned specific method, and can be carried out by any other method. For example, it is possible to measure the translation amount by a method such as quantification of the protein expression level by Western blotting, and the measurement using such a method also constitutes the present invention.

After measuring the translation amount, the cells are separated, fractionated, and selected based on, for example, the presence or absence of a protein that specifically binds to the aptamer or the difference in the expression level, and further artificial treatment or transplantation into the cells is performed. It can be used for various purposes. Such separation, sorting, and sorting are described as one aspect of the above identification. In particular, when protein-responsive mRNA is directly introduced into cells in step (1), the mRNA can be metabolized intracellularly with a half-life of about 10 hours to 1 day, which is advantageous from the viewpoint of safety. Is. According to the method for identifying an endogenous protein in a cell and the method for identifying a cell according to the present embodiment, the expression of a specific protein in a cell can be identified alive using a living cell, and further. It is very useful in that cells expressing a specific protein can be selectively separated alive.

As a further application, by using the protein-responsive mRNA according to the present embodiment in combination with the microRNA-responsive mRNA, cells expressing a specific protein and a specific microRNA are detected and / or identified. The method can be mentioned. A microRNA-responsive mRNA is an mRNA comprising a microRNA-responsive sequence in place of the aptamer described above. Such microRNA response sequences and methods for selecting live cells using these are described in detail in WO2015 / 105172, published by the present inventors.

With the protein-responsive mRNA (also referred to as mRNA switch) used in this embodiment and its production principle, the following can be expected in the future. The advantage of an mRNA switch is that the protein expressed as an output can function as an input for another mRNA switch. Therefore, by combining a plurality of mRNA switches, it is possible to construct an artificial gene circuit based on translation control and having a low risk of genomic damage. Further, by using such an artificial gene circuit, safe and highly efficient cell reprogramming (including reprogramming and differentiation) can be expected. Furthermore, by designing a circuit that takes a specific gene expression pattern, it can be expected to be applied to maintain the cell state, for example, to prevent the cultured iPS cells from spontaneously differentiating. Can be used.

Furthermore, since the translation efficiency of the invented switch correlates with the intracellular protein expression level, it is considered that the protein level in living cells can be quantified by using the protein-responsive mRNA switch. Unlike Western blotting, this method does not require cell disruption and does not require the production of a fusion protein with a detection tag, so it can be used for observing the amount of intracellular protein in a more natural state. .. In addition, by increasing the transfection efficiency, quantification of protein expression level at the single cell level can be expected. Furthermore, in combination with FACS, it is possible to isolate cells expressing a specific amount of protein.

[Second Embodiment: Structurally Stabilized LIN28A Aptamer]
According to the second embodiment, the present invention is a structurally stabilized LIN28A aptamer. The LIN28 aptamer is a stabilized LIN28A aptamer obtained by making one or more of the following modifications based on the RNA secondary structure formed by the native LIN28A aptamer sequence represented by SEQ ID NO: 1.
(1) Replace the AU base pairs constituting the stem containing the 5'-terminal base and the 3'-terminal base with GC base pairs;
(2) Replace the AU base pairs that make up the stem located between the two loops with GC base pairs; or (3) Replace the non-Watson-Crick base pairs that make up the stem located between the two loops with GC base pairs. Replace with.

The native LIN28A aptamer represented by SEQ ID NO: 1 has been reported to have a secondary structure represented by Original in FIG. 3 (Nam, Y., Chen, C., Gregory, RI, Chou, JJ). & Sliz, P. Molecular basis for interaction of let-7 microRNAs with Lin28. Cell 147, 1080-91 (2011). On the other hand, by modifying (1), 5'terminal base and 3'terminal base It is not necessary to replace all AU base pairs present in the native LIN28A aptamer with GC base pairs, as long as at least one or more AU base pairs are replaced with GC base pairs. Stabilization can be achieved, but from the perspective of more stabilization, preferably more, more preferably all AU base pairs are replaced with GC base pairs.

Next, the secondary structure represented by Original in FIG. 3 or the stabilized LIN28A aptamer modified in (1) is located between the two loops by modifying (2). The stem can be made more stable. Again, it is not necessary to replace all AU base pairs with GC base pairs, but preferably more, more preferably all AU base pairs are replaced with GC base pairs.

Further, the secondary structure represented by Original in FIG. 3, the stabilized LIN28A aptamer modified in (1), or the stabilized LIN28A aptamer modified in (2) is modified in (3). This makes it possible to further stabilize the stem located between the two loops.

In making these modifications, the base sequence important for binding to LIN28A (excluding the bases at the base of the stem that form AU base pairs or GC base pairs) is changed, or the loop It is not preferable to change the structure. This is because the loop structure is known as a structure that specifically recognizes the LIN28A protein in the natural LIN28A aptamer. Generally, in the LIN28A aptamer, the base sequences important for binding to LIN28A are GNGAY (N is an arbitrary base, Y is C or U) and GGAG, and in the specific LIN28A aptamer shown below, GGGAU and GGAG (shown underlined in the table).

Further optional modifications include (4) adding 1 to 15 base pairs to the stem containing the 5'and 3'bases.

Specific embodiments include stbA (SEQ ID NO: 2, stbA in FIG. 3), which is a stabilized LIN28A aptamer modified in (1) above, and stabilized in (1) and (2) above. There are LIN28A aptamers stbB (SEQ ID NO: 3, stbB in FIG. 3) and stbC (SEQ ID NO: 4, stbC in FIG. 3). Table 1 below shows the sequences of the native and stabilized three aptamers. Bases modified from the original aptamer are shown in lowercase.

The aptamers represented by SEQ ID NOs: 1 to 4 can be used as the aptamer sequence of protein-responsive mRNA according to the first embodiment. The design and production of protein-responsive mRNA comprising an aptamer sequence is described in detail in the first embodiment. The translational repression efficiency of the LIN28A-responsive mRNA using these aptamer sequences is, in descending order, the LIN28A-responsive mRNA having the stbC sequence, the LIN28A-responsive mRNA having the stbB sequence, and the LIN28A-responsive mRNA having the stbA sequence. It is a LIN28A responsive mRNA having a native LIN28A aptamer sequence.

The stabilized LIN28A aptamer according to the second embodiment is useful for detecting specific expression of LIN28A protein, and particularly useful for detecting pluripotent stem cells such as iPS cells and ES cells. In addition, LIN28A-responsive mRNA (also referred to as LIN28A switch) can be expected to be used as these stem cell selection techniques. Since LIN28A is a protein present in the cytoplasm, there is an advantage that the intracellular protein can be easily detected by an mRNA switch such as LIN28A-responsive mRNA as long as RNA enters the cytoplasm. By changing the protein expressed by the switch from a reporter protein to an apoptosis-inducing protein, cell death is induced in a non-stem cell-specific manner. As described above, the use of protein-responsive mRNA switches has a low risk of genomic damage, and therefore can be expected to be applied to the field of regenerative medicine as a safe stem cell / differentiated cell selection technique. Therefore, the development of new nucleic acid drugs with few side effects can be expected.

[Third Embodiment: Modified U1A aptamer]
According to the third embodiment, the present invention is a structurally stabilized U1A aptamer. The U1A aptamer is a structurally stabilized U1A aptamer obtained by making the following modifications based on the RNA secondary structure formed by the natural U1A aptamer sequence represented by SEQ ID NO: 6.
(1) Add 1 to 15 AU base pairs or GC base pairs to a stem structure containing 5'-terminal bases and 3'-terminal bases. Alternatively, 1 to 15 AU base pairs or GC base pairs are added to the stem existing between the two boxes. Through the above modifications, the aptamer is designed to form the secondary structure required for binding to the target.
Alternatively, it is a structure-stabilized U1A aptamer based on the RNA secondary structure formed by the U1A aptamer sequence represented by SEQ ID NO: 8 and with the following modifications.
(2) Add 1 to 15 AU base pairs or GC base pairs to a stem containing 5'-terminal bases and 3'-terminal bases.

It has been reported that the natural U1A aptamer represented by SEQ ID NO: 6 has a secondary structure represented by the lower part of U1utr in FIG. 12 (Reference [4]). On the other hand, by modifying (1), the stem existing between Box 1 and Box 2 can be stabilized. The base pairs of 1 to 15 to be added may be AU base pairs, GC base pairs, or a combination thereof, but preferably, more GC base pairs are added. More preferably, all added base pairs are GC base pairs.

As a specific embodiment, there is s U1utr_stb (SEQ ID NO: 7, U1utr_stb in FIG. 12) which is a stabilized U1A aptamer obtained by modifying the above (1). This sequence and the sequence of the native U1utr are shown in Table 2 below.

It has been reported that the document-known U1A aptamer represented by SEQ ID NO: 8 has a secondary structure represented by U1LSL in FIG. 12 (Reference [5]). On the other hand, by modifying (2), the stem containing the 5'-terminal base and the 3'-terminal base can be stabilized. The base pairs of 1 to 15 to be added may be AU base pairs, GC base pairs, or a combination thereof, but preferably, more GC base pairs are added. More preferably, all added base pairs are GC base pairs.

Specific embodiments include U1LSL + 10bp (SEQ ID NO: 9, U1LSL + 10bp in FIG. 12) and U1LSL + 15bp (SEQ ID NO: 10, FIG. 12), which are stabilized U1A aptamers modified in (2) above. There is U1LSL + 15bp). These sequences and the sequences of the native U1LSL are shown in Table 2 below.

The aptamer represented by SEQ ID NOs: 5 to 10 and the stabilized aptamer can be used as the aptamer sequence of the protein-responsive mRNA according to the first embodiment. The design and production of protein-responsive mRNA comprising an aptamer sequence is described in detail in the first embodiment.

Stabilized U1A aptamers according to the third embodiment may be useful for detecting specific expression of U1A protein. It has been reported that U1A protein is highly expressed in certain types of cancer cells, and by applying the method for detecting intracellular endogenous proteins based on the first embodiment, cancer cells can be detected and sorted. There is expected.

[Fourth Embodiment: Modified p50 aptamer]
The present invention is a structurally stabilized p50 aptamer according to a fourth embodiment. The p50 aptamer is an artificially obtained aptamer represented by SEQ ID NO: 83, and based on the RNA secondary structure formed by the artificial p50 aptamer reported in Ref. [6], the following modifications:
(1) A structure-stabilized p50 aptamer obtained by adding 1 to 20 AU base pairs or GC base pairs to a stem structure containing 5'-terminal bases and 3'-terminal bases.

It has been reported that the p50 aptamer represented by SEQ ID NO: 83 has the secondary structure shown in the left figure of FIG. 17 (Reference [6]). On the other hand, the stem can be stabilized by modifying (1). The 1 to 20 base pairs to be added may be AU base pairs, GC base pairs, or a combination thereof, but preferably, more GC base pairs are added. More preferably, all added base pairs are GC base pairs. The base pair to be added is preferably 5 to 20 base pairs, and more preferably 10 to 20 base pairs.

As a specific embodiment, there is p50A-stb (SEQ ID NO: 84, p50A-stb in the right figure of FIG. 17) which is a stabilized p50 aptamer obtained by modifying the above (1). This sequence and the sequence of the native U1utr are shown in Table 3 below.

The aptamer represented by SEQ ID NO: 84 and the stabilized aptamer can be used as an aptamer sequence in the protein-responsive mRNA described in the first embodiment. The design and production of protein-responsive mRNA comprising an aptamer sequence is described in detail in the first embodiment.

The stabilized p50 aptamer according to the fourth embodiment may be useful for detecting the specific expression of the cancer-related protein p50. It has been reported that p50 is highly expressed in certain types of cancer cells, and can be used for detecting and sorting cancer cells by applying the method for detecting intracellular proteins based on the first embodiment. Be expected.

[Fifth Embodiment: Method for identifying cell differentiation (reprogramming) state]
According to the fifth embodiment, the present invention is a method for identifying a differentiated state of cells. The method includes the following steps.
(1) Introduce an mRNA containing an aptamer sequence specific to a protein expressed by an undifferentiated cell and a marker gene sequence functionally linked to the aptamer sequence, or a vector encoding the mRNA into a cell group that can contain pluripotent stem cells. Steps; and (2) A step of identifying the differentiation state of cells based on the amount of translation of the marker gene.

In the present embodiment, "identification of the differentiated state of a cell" means to identify that a cell is in a differentiated or undifferentiated state, its change, degree, and the like. For example, in one embodiment (a), cells contacted with a reprogramming factor are cultured for a predetermined method and period and then reprogrammed into induced pluripotent stem (iPS) cell states. It means to detect, detect, recognize, and identify the state related to initialization, such as whether the reprogramming is not done well and the iPS cell is not the target iPS cell, or it also includes separation and separation. Shall be. It is known from previous studies that iPS cells express LIN28A (Tano, K. et al. A Novel In Vitro Method for Detecting Undifferentiated Human Pluripotent Stem Cells as Impurities in Cell Therapy Products Using a Highly Efficient). Culture System. PLoS One 9, e110496 (2014).) Among the cells contacted with the reprogramming factor, cells expressing a sufficient amount of LIN28A (cells having endogenous LIN28A) are in the desired reprogramming state. It can be said that it is an iPS cell for the purpose of having. Further, in another embodiment (b), pluripotent stem cells including iPS cells are detected as pluripotent stem cells remaining without differentiation in a cell group subjected to a predetermined differentiation operation. However, it means to detect, recognize, or selectively separate and segregate those cells. When iPS cells and other pluripotent stem cells are differentiated into desired cells for clinical use, the most impediment is the cells that remain undifferentiated and have the ability to form tumors (PLoS One). . 2012; 7 (5): e37342. Doi: 10.1371 / journal.pone.0037342. Epub 2012 May 17.). Therefore, a technique for selectively removing pluripotent cells from the differentiated cell group has been strongly desired. In the present invention, cells retaining pluripotency are detected by using an aptamer sequence specific to a protein expressed by undifferentiated cells, typically a LIN28A-responsive mRNA that specifically detects LIN28A. It can be detected and recognized, and in a preferred embodiment, it can be separated and selectively removed. At this time, it has been confirmed that the LIN28A switch can identify undifferentiated cells without significant side effects. However, the identification of the differentiated state of cells is not limited to a specific embodiment, and undifferentiated cells are detected, identified, separated, and removed from a group of cells which can be a mixture of cells having different differentiated states. Make it possible.

The "cell group capable of containing pluripotent stem cells" used in the step (1) of the present embodiment is, for example, cells that have been contacted with a reprogramming factor, and after being cultured for a predetermined method and period, the cells are reprogrammed. It may be a cell that is programmed and predicted to be in the state of induced pluripotent stem (iPS) cells. Alternatively, it is a group of cells in which pluripotent stem cells have been subjected to a predetermined differentiation operation, and undifferentiated cells can remain. However, it may be a group of cells that does not contain undifferentiated cells. Alternatively, it may be a cell group that can contain any other pluripotent stem cell, and the purpose of detection and the origin of the cell group are not particularly limited.

Next, cells contacted with a reprogramming factor, which is a group of cells that can contain pluripotent stem cells preferably used in embodiment (a), will be described. In the present embodiment, the cell contacted with the reprogramming factor typically refers to an artificially engineered cell for the purpose of preparing iPS cells. iPS cells can be produced by introducing specific reprogramming factors into somatic cells in the form of DNA, RNA or protein, with properties similar to ES cells, such as pluripotency and self-renewal proliferation. It is an artificial stem cell derived from a somatic cell having the ability (K. Takahashi and S. Yamanaka (2006) Cell, 126: 663-676; K. Takahashi et al. (2007), Cell, 131: 861-872; J. Yu et al. (2007), Science, 318: 1917-1920; Nakagawa, M. et al., Nat. Biotechnol. 26: 101-106 (2008); International Publication WO 2007/069666). Reprogramming factors are genes that are specifically expressed in ES cells, their gene products or non-cording RNAs, or genes that play an important role in maintaining undifferentiated ES cells, their gene products or non-coding RNAs, or It may be composed of low molecular weight compounds. Genes included in the reprogramming factors include, for example, Oct3 / 4, Sox2, Sox1, Sox3, Sox15, Sox17, Klf4, Klf2, c-Myc, N-Myc, L-Myc, Nanog, Lin28, Fbx15, ERas, ECAT15. -2, Tcl1, beta-catenin, Lin28b, Sall1, Sall4, Esrrb, Nr5a2, Tbx3, Glis1, etc. are exemplified, and these initialization factors may be used alone or in combination. The combinations of reprogramming factors include WO2007 / 069666, WO2008 / 118820, WO2009 / 007852, WO2009 / 032194, WO2009 / 058413, WO2009 / 057831, WO2009 / 075119, WO2009 / 079007, WO2009 / 091659, WO2009 / 101084, WO2009 / 101407, WO2009 / 102983, WO2009 / 114949, WO2009 / 117439, WO2009 / 126250, WO2009 / 126251, WO2009 / 126655, WO2009 / 157593, WO2010 / 009015, WO2010 / 033906, WO2010 / 033920, WO2010 / 042800, WO2010 / 050626, WO 2010/056831, WO2010 / 068955, WO2010 / 098419, WO2010/102267, WO 2010/111409, WO 2010/111422, WO2010 / 115050, WO2010 / 124290, WO2010 / 147395, WO2010 / 147612, Huangfu D, et al. ( 2008), Nat. Biotechnol., 26: 795-797, Shi Y, et al. (2008), Cell Stem Cell, 2: 525-528, Eminli S, et al. (2008), Stem Cells. 26: 2467 -2474, Huangfu D, et al. (2008), Nat Biotechnol. 26: 1269-1275, Shi Y, et al. (2008), Cell Stem Cell, 3, 568-574, Zhao Y, et al. (2008) ), Cell Stem Cell, 3: 475-479, Marson A, (2008), Cell Stem Cell, 3, 132-135, Feng B, et al. (2009), Nat Cell Biol. 11: 197-203, RL Judson et al., (2009), Nat. Biotech., 27: 459-461, Lyssiotis CA, et al. (2009), Proc Natl Acad Sci US A. 106: 8912 -8917, Kim JB, et al. (2009), Nature. 461: 649-643, Ichida JK, et al. (2009), Cell Stem Cell. 5: 491-503, Heng JC, et al. (2010) , Cell Stem Cell. 6: 167-74, Han J, et al. (2010), Nature. 463: 1096-100, Mali P, et al. (2010), Stem Cells. 28: 713-720, Maekawa M , Et al. (2011), Nature. 474: 225-9.

The reprogramming factors include histone deacetylase (HDAC) inhibitors [eg, small molecule inhibitors such as valproic acid (VPA), tricostatin A, sodium butyrate, MC 1293, M344, siRNA and shRNA for HDAC (eg). , HDAC1 siRNA Smartpool® (Millipore), HuSH 29mer shRNA Constructs against HDAC1 (OriGene), etc.), MEK inhibitors (eg PD184352, PD98059, U0126, SL327 and PD0325901), Glycogen Small molecule inhibitors such as synthase kinase-3 inhibitors (eg Bio and CHIR99021), DNA methyltransferase inhibitors (eg 5-azacytidine), histone methyltransferase inhibitors (eg BIX-01294), Suv39hl, Suv39h2, SetDBl And nucleic acid expression inhibitors such as siRNA and shRNA against G9a), L-channel calcium agonist (eg Bayk8644), butyric acid, TGFβ inhibitor or ALK5 inhibitor (eg LY364947, SB431542, 616453 and A-83-01) , P53 inhibitors (eg siRNA and shRNA for p53), ARID3A inhibitors (eg siRNA and shRNA for ARID3A), miRNAs such as miR-291-3p, miR-294, miR-295 and mir-302, Wnt Signaling ( For example, for the purpose of increasing the establishment efficiency of soluble Wnt3a), neuropeptide Y, prostaglandins (eg, prostaglandins E2 and prostaglandins J2), hTERT, SV40LT, UTF1, IRX6, GLISl, PITX2, DMRTBl, etc. Factors used are also included, and in the present specification, these factors used for the purpose of improving establishment efficiency are not particularly distinguished from reprogramming factors.

In the case of protein form, the reprogramming factor may be introduced into somatic cells by techniques such as lipofection, fusion with cell membrane penetrating peptides (eg, HIV-derived TAT and polyarginine), and microinjection.

On the other hand, in the case of DNA morphology, it can be introduced into somatic cells by a method such as a virus, a plasmid, a vector such as an artificial chromosome, lipofection, liposome, or microinjection. Viral vectors include retroviral vectors and lentiviral vectors (Cell, 126, pp.663-676, 2006; Cell, 131, pp.861-872, 2007; Science, 318, pp.1917-1920, 2007. ), Adenovirus vector (Science, 322, 945-949, 2008), adeno-associated virus vector, Sendai virus vector (WO 2010/008054) and the like. Further, the artificial chromosome vector includes, for example, a human artificial chromosome (HAC), a yeast artificial chromosome (YAC), a bacterial artificial chromosome (BAC, PAC) and the like. As the plasmid, a plasmid for mammalian cells can be used (Science, 322: 949-953, 2008). The vector can contain regulatory sequences such as promoters, enhancers, ribosome binding sequences, terminators, polyadenylation sites, etc. so that nuclear reprogramming substances can be expressed, and, if desired, drug resistance genes ( For example, canamycin resistance gene, ampicillin resistance gene, puromycin resistance gene, etc.), thymidin kinase gene, diphtheriatoxin gene, and other selectable marker sequences, green fluorescent protein (GFP), β-glucuronidase (GUS), FLAG, and other reporter gene sequences. Can include. In addition, the above vector has LoxP sequences before and after the gene encoding the reprogramming factor or the promoter and the gene encoding the reprogramming factor that binds to the promoter after introduction into somatic cells. You may.

In the case of RNA form, it may be introduced into somatic cells by a method such as lipofection or microinjection, and in order to suppress degradation, RNA incorporating 5-methylcytidine and pseudouridine (TriLink Biotechnologies) is used. May be (Warren L, (2010) Cell Stem Cell. 7: 618-630).

Cultures for iPS cell induction include, for example, DMEM, DMEM / F12 or DME cultures containing 10-15% FBS (these cultures also include LIF, penicillin / streptomycin, puromycin, L- Glutamine, non-essential amino acids, β-mercaptoethanol, etc. can be appropriately contained) or a commercially available culture medium [for example, mouse ES cell culture medium (TX-WES culture medium, Thrombo X), primate ES. Culture medium for cell culture (culture solution for primate ES / iPS cells, Reprocell), serum-free medium (mTeSR, Stemcell Technology, Essential 8, Life Technologies, StemFit, Ajinomoto)] and the like are included.

As an example of the culture method, for example, in the _{presence of 5% CO 2} at 37 ° C., the somatic cells are brought into contact with the reprogramming factor on DMEM or DMEM / F12 culture medium containing 10% FBS and cultured for about 4 to 7 days. Then, the cells are re-sown on feeder cells (for example, mitomycin C-treated STO cells, SNL cells, etc.), and about 10 days after contact between the somatic cells and the reprogramming factor, the cells are used in a culture medium for bFGF-containing primate ES cell culture. It can be cultured to give rise to iPS-like colonies about 30-about 45 days or more after the contact.

Alternatively, in the _{presence of 5% CO 2} at 37 ° C., DMEM culture medium containing 10% FBS (for example, LIF, penicillin / streptomycin, etc.) on feeder cells (eg, mitomycin C-treated STO cells, SNL cells, etc.). It can appropriately contain puromycin, L-glutamine, non-essential amino acids, β-mercaptoethanol, etc.), and ES-like colonies can be generated after about 25 to about 30 days or more. .. Desirably, instead of feeder cells, the reprogrammed somatic cells themselves are used (Takahashi K, et al. (2009), PLoS One. 4: e8067 or WO2010 / 137746), or extracellular matrix (eg, Laminin-). 5 (WO2009 / 123349), Matrigel (BD) and iMatrix511 (Nippi)) are exemplified.

In addition to this, a method of culturing using a medium containing no serum is also exemplified (Sun N, et al. (2009), Proc Natl Acad Sci US A. 106: 15720-15725). Furthermore, in order to increase the efficiency of establishment, iPS cells may be established under hypoxic conditions (oxygen concentration of 0.1% or more and 15% or less) (Yoshida Y, et al. (2009), Cell Stem Cell. 5: 237. -241 or WO2010 / 013845).

During the above culture, fresh culture solution and culture solution are exchanged once a day from the second day after the start of culture. The number of somatic cells used for nuclear reprogramming is not limited, but ranges from about 5 × 10 ³ to about 5 × 10 ⁶ cells ^{per 100 cm 2 culture dish.} As used herein, the term "somatic cell" refers to any animal cell (preferably a mammalian cell, including human) except germline cells such as eggs, oocytes, ES cells or totipotent cells. Say. Somatic cells include, but are not limited to, fetal (pup) somatic cells, neonatal (pup) somatic cells, and mature healthy or diseased somatic cells, as well as primary cultured cells. , Passed cells, and established cells are all included. Specifically, somatic cells include, for example, (1) tissue stem cells (somatic stem cells) such as nerve stem cells, hematopoietic stem cells, mesenchymal stem cells, and dental pulp stem cells, (2) tissue precursor cells, (3) lymphocytes, and epithelium. Cells, endothelial cells, muscle cells, fibroblasts (skin cells, etc.), hair cells, hepatocytes, gastric mucosal cells, intestinal cells, splenocytes, pancreatic cells (pancreatic exocrine cells, etc.), brain cells, lung cells, renal cells And differentiated cells such as fat cells are exemplified. When iPS cells are used as a material for transplant cells, it is desirable to use somatic cells having the same or substantially the same HLA genotype of the transplanted individual from the viewpoint of not causing rejection. Here, "substantially the same" means that the transplanted cells have the same HLA genotype to the extent that the immunosuppressant can suppress the immune response. For example, HLA-A and HLA-B. It is a somatic cell having an HLA type in which 3 loci of HLA-DR or 4 loci with HLA-C are matched.

Next, a cell group in which a predetermined differentiation operation has been performed on the pluripotent stem cells, which is a cell group that can contain pluripotent stem cells preferably used in the embodiment (b), will be described. In the present invention, a pluripotent stem cell is a stem cell having pluripotency capable of differentiating into many cells existing in a living body and also having a proliferative ability, and is arbitrarily induced into a desired differentiated cell. Cells are included. The pluripotent stem cells are not particularly limited, and are, for example, embryonic stem (ES) cells, embryonic stem (ntES) cells derived from cloned embryos obtained by nuclear transplantation, sperm stem cells (“GS cells”), and embryonic stem cells. Includes germ cells (“EG cells”), induced pluripotent stem (iPS) cells, cultured fibroblasts and pluripotent cells derived from bone marrow stem cells (Muse cells). Preferred pluripotent stem cells are iPS cells, more preferably human iPS cells, from the viewpoint that they can be obtained without destroying embryos, eggs, etc. in the production process. Other pluripotent stem cells in addition to the above iPS cells will be described below.

(A) Embryonic stem cells Embryonic stem cells (ES cells) have pluripotency and self-renewal proliferative capacity established from the inner cell mass of early embryos (eg, blastocysts) of mammals such as humans and mice. It is a stem cell that has.

ES cells are embryo-derived stem cells derived from the inner cell mass of the blastocyst, which is the embryo after the morula at the 8-cell stage of the fertilized egg, and have the ability to differentiate into all the cells that make up the adult, so-called multi-differentiation. It has the ability and the ability to proliferate by self-replication. ES cells were discovered in mice in 1981 (MJ Evans and MH Kaufman (1981), Nature 292: 154-156), and ES cell lines were subsequently established in primates such as humans and monkeys (JA Thomson et. al. (1998), Science 282: 1145-1147; JA Thomson et al. (1995), Proc. Natl. Acad. Sci. USA, 92: 7844-7848; JA Thomson et al. (1996), Biol. Reprod ., 55: 254-259; JA Thomson and VS Marshall (1998), Curr. Top. Dev. Biol., 38: 133-165).

ES cells can be established by removing the inner cell mass from the blastocyst of the fertilized egg of the target animal and culturing the inner cell mass on a fibroblast feeder. For cell maintenance by subculture, use a culture medium supplemented with substances such as leukemia inhibitory factor (LIF) and basic fibroblast growth factor (bFGF). It can be carried out. For methods of establishing and maintaining ES cells in humans and monkeys, see, for example, USP 5,843,780; Thomson JA, et al. (1995), Proc Natl. Acad. Sci. US A. 92: 7844-7848; Thomson JA, et. al. (1998), Science. 282: 1145-1147; H. Suemori et al. (2006), Biochem. Biophys. Res. Commun., 345: 926-932; M. Ueno et al. (2006), Proc . Natl. Acad. Sci. USA, 103: 9554-9559; H. Suemori et al. (2001), Dev. Dyn., 222: 273-279; H. Kawasaki et al. (2002), Proc. Natl. Acad. Sci. USA, 99: 1580-1585; Klimanskaya I, et al. (2006), Nature. 444: 481-485, etc.

As the culture medium for ES cell production, for example, DMEM / F-12 culture medium supplemented with 0.1 mM 2-mercaptoethanol, 0.1 mM non-essential amino acid, 2 mM L-glutamic acid, 20% KSR and 4 ng / ml bFGF was used. , 37 ° C, 5% CO ₂ , human ES cells can be maintained in a moist atmosphere (H. Suemori et al. (2006), Biochem. Biophys. Res. Commun., 345: 926-932). Also, ES cells need to be passaged every 3-4 days, where passage is 0.25% trypsin and 0.1 mg / ml collagenase IV in PBS containing _{, for example, 1 mM CaCl 2 and 20% KSR.} Can be done using.

ES cells can generally be selected by the Real-Time PCR method using the expression of gene markers such as alkaline phosphatase, Oct-3 / 4, and Nanog as an index. In particular, in the selection of human ES cells, the expression of genetic markers such as OCT-3 / 4, NANOG, and ECAD can be used as an index (E. Kroon et al. (2008), Nat. Biotechnol., 26: 443). -452).

Human ES cell lines, for example, WA01 (H1) and WA09 (H9) are obtained from the WiCell Research Institute, and KhES-1, KhES-2 and KhES-3 are obtained from the Institute for Frontier Medical Sciences, Kyoto University (Kyoto, Japan). It is possible.

(B) Sperm stem cells Sperm stem cells are testis-derived pluripotent stem cells, which are the origin cells for spermatogenesis. Similar to ES cells, these cells can induce differentiation into cells of various lineages, and have properties such as the ability to produce chimeric mice when transplanted into mouse blastocysts (M. Kanatsu-Shinohara et al. (M. Kanatsu-Shinohara et al.). 2003) Biol. Reprod., 69: 612-616; K. Shinohara et al. (2004), Cell, 119: 1001-1012). It is self-renewable in a culture medium containing glial cell line-derived neurotrophic factor (GDNF), and sperm by repeating passage under the same culture conditions as ES cells. Stem cells can be obtained (Masanori Takebayashi et al. (2008), Experimental Medicine, Vol. 26, No. 5 (Special Edition), pp. 41-46, Yodosha (Tokyo, Japan)).

(C) Embryonic germ cells Embryonic germ cells are cells that are established from primordial germ cells in the embryonic period and have pluripotency similar to ES cells, such as LIF, bFGF, and stem cell factor. It can be established by culturing primordial germ cells in the presence of the substance of (Y. Matsui et al. (1992), Cell, 70: 841-847; JL Resnick et al. (1992), Nature, 359: 550. -551).

(D) ES cells derived from cloned embryos obtained by nuclear transplantation ES cells derived from cloned embryos (nt ES cells) obtained by nuclear transplantation are ES cells derived from cloned embryos produced by nuclear transplantation technology. It has almost the same characteristics as ES cells derived from fertilized eggs (T. Wakayama et al. (2001), Science, 292: 740-743; S. Wakayama et al. (2005), Biol. Reprod., 72. : 932-936; J. Byrne et al. (2007), Nature, 450: 497-502). That is, ES cells established from the inner cell mass of blastocysts derived from cloned embryos obtained by replacing the nuclei of unfertilized eggs with the nuclei of somatic cells are nt ES (nuclear transfer ES) cells. For the production of nt ES cells, a combination of nuclear transfer technology (JB Cibelli et al. (1998), Nature Biotechnol., 16: 642-646) and ES cell production technology is used (Kiyoka Wakayama et al. (Wakayama et al.) 2008), Experimental Medicine, Vol. 26, No. 5 (Special Edition), pp. 47-52). In nuclear transfer, somatic cell nuclei can be injected into unfertilized mammalian eggs and cultured for several hours to reprogram them.

(E) Multilineage-differentiating Stress Enduring cells
Multilineage-differentiating Stress Enduring cells (Muse cells) are pluripotent stem cells produced by the method described in WO2011 / 007900, specifically, fibroblasts or bone marrow stromal cells treated with long-term trypsin, It is a pluripotent cell obtained by suspension culture after treatment with trypsin for 8 hours or 16 hours, and is positive for SSEA-3 and CD105.

Methods for inducing differentiation of these pluripotent stem cells into predetermined differentiated cells are known, and those skilled in the art can carry out specific differentiation-inducing methods according to the cells of interest. As an example, cardiomyocytes can be produced from pluripotent stem cells as a method for inducing differentiation into cardiomyocytes, for example, by the method reported by Laflamme MA et al. (Laflamme MA & Murry CE, Nature 2011, Review). In addition to this, although not particularly specified, for example, a method of producing cardiomyocytes by forming a cell mass (embryonic body) by suspension culture of induced pluripotent stem cells, cardiomyocytes in the presence of a substance that suppresses BMP signal transduction. Method for producing cells (WO2005 / 033298), method for producing cardiomyocytes by adding Activin A and BMP in order (WO2007 / 002136), manufacturing cardiomyocytes in the presence of substances that promote activation of canonical Wnt signaling pathway (WO2007 / 126077) and a method of isolating Flk / KDR-positive cells from induced pluripotent stem cells and producing cardiomyocytes in the presence of cyclosporin A (WO2009 / 118928) are exemplified. As another example, as a method for inducing differentiation into histiocytes such as endothelial cells, hepatocytes or insulin-producing cells, for example, Kajiwara M, et al, Proc Natl Acad Sci US A. 109: 12538-12543, 2012, Kunisada Y , Et al, Stem Cell Res. 8: 274-284, 2012, Nakagawa, M, et al, Sci Rep 4, 3594, 2014, etc., to produce histiocytes from pluripotent hepatocytes. it can. As yet another example, as a method for inducing differentiation into nerve cells, nerve cells can be produced from pluripotent stem cells by, for example, the methods disclosed in WO2011 / 019092 and WO2011 / 158960.

Step (1) is a step of introducing a vector encoding mRNA having an aptamer sequence specific to a protein expressed by undifferentiated cells into a cell group that can contain pluripotent stem cells such as the cell group exemplified above. is there. The step of introducing the mRNA or vector into the cell may be the same as the method described in the step (1) of the first embodiment. This step may be performed, for example, for embodiments (a) and (b) at any time during the initialization step or the differentiation step, in a reprogramming state or at any time. A differentiated state can be obtained. The embodiment (a) can be carried out particularly at a time when iPS-like colonies are formed and iPS cells are predicted to be generated, that is, at a time when it is predicted to be reprogrammed. At this point, the culture medium for iPS cells contains a mixture of reprogrammed iPS cells and non-reprogrammed cells, which has the advantage of identifying the reprogrammed iPS cells. The embodiment (b) can be carried out at a time when it is predicted that a predetermined differentiation-induced cell, for example, a cardiomyocyte or a nerve cell is obtained.

The step (2) is a step of identifying the differentiation state of the cell based on the translation amount of the marker gene. The identification step can be carried out in a manner substantially similar to that described in the first embodiment. For example, by measuring the translational repression efficiency of a marker gene, the expression level of a protein expressed in an undifferentiated cell, typically LIN28A in the cell, can be quantified. Then, it is possible to obtain information on whether or not the cell is a reprogrammed iPS cell or another pluripotent stem cell (undifferentiated cell). The iPS cells reprogrammed to the desired state and the undesired undifferentiated cells remaining in the differentiated cells can be selectively separated after or at the same time as the differentiation. These separations can be carried out by using a fluorescent protein as a marker gene and separating cells in a predetermined translation state by flow cytometry or imaging cytometry. Alternatively, when a drug-resistant protein or a lethal gene is used as the marker protein, it is possible to carry out selective isolation at the same time as identification.

Conventionally, pluripotent stem cells such as iPS cells and ES cells can be selected according to the shape of the formed colonies. In addition, cell surface proteins expressed when somatic cells are reprogrammed (eg, SSEA-1, SSEA-3, SSEA-4, TRA-2-54, TRA-1-60 and TRA-1- 80) was selected as an index. According to the method for detecting the differentiated state and the reprogramming state of cells according to the fifth embodiment of the present invention, an mRNA having an aptamer sequence specific to a protein expressed by an undifferentiated cell, particularly a LIN28A responsive mRNA, is used. Using the expression of proteins expressed by undifferentiated cells, especially LIN28A, as an index, it is possible to detect the differentiated state and reprogramming state of cells alive and selectively separate and separate iPS cells or undifferentiated cells. Become. Compared with the conventional method, there are advantages that the intracellular gene expression state can be detected alive and the intracellular information can be visualized alive, in particular, instead of the cell surface protein.

Hereinafter, the present invention will be described in more detail with reference to examples. The following examples do not limit the present invention.

[Plasid construction]
First, pTAPmyc-T2A-tagRFP and pAptamerCassette-EGFP were prepared as empty vectors for the trigger plasmid and the switch plasmid, respectively.
In the preparation of pTAPmyc -T2A-tagRFP, specifically, first, pIRES2-DsRed Express (Clontech) was cleaved with BamHI and NheI, and then double-stranded DNA obtained by annealing synthetic oligos SKW003 and SKW004 was inserted, and pTAP- IRES2-DsRed Ex was prepared. Furthermore, this plasmid and pTAP-myc were cleaved with BamHI and XbaI and inserted into pTAP-IRES2-DsRed Ex to prepare pTAPmyc-IRES2-DsRed Ex. Next, the target moiety was PCR-amplified using pTAP-BS15-T2A-tagRFP as a template and synthetic oligos KWC0093 and KWC0094 as a primer set. This PCR product and pTAPmyc-IRES2-DsRed Ex were cleaved with BglII and NotI, and the digested PCR product was inserted to obtain pTAPmyc -T2A-tagRFP.

pAptamerCassette-EGFP was prepared by cleaving pBoxCDGC-kMet-EGFP (reference [1]) with NheI and AgeI and then inserting double-stranded DNA obtained by annealing synthetic oligos SKC0041 and SKC0042. In the preparation of the LIN28A expression plasmid (pLIN28Amyc-T2A-tagRFP), the target portion was first amplified using pTrg5H-hLin28 as a template and synthetic oligos SKC0052 and KWC0053 as a primer set. This PCR product and pTAPmyc-T2A-tagRFP were cleaved with SalI and BamHI, and then both were ligated to obtain pLIN28Amyc-T2A-tagRFP.

In the preparation of the U1A expression plasmid (pu1Amyc-T2A-tagRFP), the target portion was first amplified using pTrg5H-U1A as a template and synthetic oligos SKC0052 and KWC0053 as a primer set. This PCR product and pTAPmyc-T2A-tagRFP were cleaved with SalI and BamHI, and then both were ligated to obtain pU1Amyc-T2A-tagRFP.
The sequences of the synthetic oligo DNAs used so far are shown in Table 4 below.

In the preparation of each switch plasmid, first, pAptamerCassette-EGFP was cleaved with AgeI and BamHI. Next, double-stranded DNA obtained by annealing the synthetic oligo with the combination shown in Table 5 below was prepared and inserted into the digested pAptamerCassette-EGFP to obtain each LIN28A switch plasmid.

Each U1A switch plasmid was prepared in the same manner as the LIN28A switch using oligos in the combinations shown in Table 6.

培養細胞へのトランスフェクションのためのプラスミドは、すべてJETSTAR 2.0 (VERITAS)を用いて大量精製した。

All plasmids for transfection into cultured cells were mass purified using JETSTAR 2.0 (VERITAS).

[Building IVT template]
The 5'UTR and 3'UTR sequences for protein coding region (ORF), trigger mRNA and reference mRNA production were PCR amplified from plasmid or oligo DNA using appropriate primers. The plasmids used as templates and the primers used, and the oligo DNAs used as templates and the primers used are shown in Tables 7, 8 and 9 below, respectively.

To generate IVT templates for trigger mRNA and reference mRNA synthesis, 5'UTR fragments, 3'UTR fragments, and ORFs were PCR amplified and ligated using the T7FwdA and Rev120A primer sets in Table 10.

To generate an IVT template for switch mRNA synthesis, 3'UTR fragments, spacer sequences and ORFs were PCR amplified and ligated using 5'UTR primers and Rev120A primer sets. Table 11 shows the 5'UTR primer and spacer sequences used to prepare each switch mRNA.

PCR products were purified using the MinElute PCR purification kit (QIAGEN) according to the manufacturer's instructions. If the PCR product was amplified from the plasmid prior to purification, it was digested with Dpn I (TOYOBO) at 37 ° C for 30 minutes.

[Synthesis and purification of mRNA]
mRNA synthesis was adjusted using the MegaScript T7 kit (Ambion) according to reference [2]. In this reaction, except for switch mRNA, pseudouridine-5'-triphosphate and 5-methylcytidine-5'-triphosphate (TriLink Bio Technologies) were used in place of uridine triphosphate and cytidine triphosphate, respectively. did. The guanosine triphosphate used was diluted 5-fold with Anti-Reverse Cap Analog (New England Biolabs). The reaction mixture was reacted at 37 ° C for 6 hours, TURBO DNase (Ambion) was added, and the mixture was further subjected to constant temperature treatment at 37 ° C for 30 minutes. The obtained mRNA was purified by FavorPrep Blood / Cultured Cell total RNA extraction clumn (Favorgen Biotech) and treated at 37 ° C for 30 minutes using Antarctic Phosphatase (New England Biolabs). Then, it was further purified by RNeasy MinElute Cleanup Kit (QIAGEN).

[Cell culture]
HeLa cells were cultured in Dulbecco's Modified Eagle's Medium (DMEM) -F12 (Invitrogen) containing 10% Fetal Bovine Serum (FBS). HEK293FT cells were cultured in DMEM (Nakalitex) supplemented with 10% FBS, 2 mM L-Glutamine (Invitrogen), 0.1 mM Non-Essential Amino Acids (Invitrogen), and 1 mM Sodium Pyruvate (Sigma). iPS cells were cultured on a laminin-coated 6-well plate using StemFit (Ajinomoto) under feeder-free conditions.

[Plasmid transfection]
The cultured cells were seeded on a 24-well plate, and the purified plasmid was introduced the next day to a total volume of 500 ng. In experiments with mutants, HEK293FT cells were seeded in 96-well plates and the next day purified plasmid was introduced to a total volume of 125 ng. The introduction was carried out using 2 μL of Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions. The plasmids used were mixed in Opti-MEM (Invitrogen) so that the volume ratio of the switch plasmid and the trigger plasmid was 1: 4. The medium was changed 4 to 6 hours after transfection.

[MRNA transfection]
The cultured cells were seeded on a 24-well plate, and the synthesized mRNA was introduced the next day. The introduction was carried out using 1 μL of StmFect (Stemgent) according to the manufacturer's instructions. In experiments using HEK293FT cells, 100 ng of switch mRNA, 0 to 300 ng of trigger mRNA, and 50 ng of reference mRNA were co-introduced. In experiments using iPS cells, 100 ng of switch mRNA and 100 ng of reference mRNA were co-introduced. The medium was changed 4 to 6 hours after transfection. For transfection of iPS cells, the medium was changed at least 1 hour before introduction.

[Flow cytometry]
The day after transfection, cells were separated from the plate, passed through a mesh and analyzed by flow cytometry. In experiments with 24-well plates, Accuri C6 (BD Biosciences) was used. EGFP and hmAG1 were detected by the FL1 (530/30 nm) filters fitter. tagRFP and mKO2 were detected by FL2 (585/40 nm) filters fitter. iRFP670 was detected by FL4 (675 / 12.5 nm) filters fitter. BD LSRFORTESSA (BD Bioscuence) was used in experiments using 96-well plates. EGFP and tagRFP were analyzed by a Blue laser equipped with a FITC filter (530/30 nm) and a Green laser equipped with a PE filter (585/42 nm), respectively. Dead cells and debris were excluded by anterior and lateral light scattering signals. For the calculation of translation efficiency using a plasmid, among living cells, those having a fluorescence value of tagRFP above a certain level were analyzed. For the calculation of translation efficiency using mRNA, among living cells, those having a fluorescence value of iRFP670 or mKO2 above a certain level were analyzed.

[Induction and transfection of LIN28A expression by doxycycline] Cell lines capable of inducing LIN28A expression by addition of doxicycline are 10% FBS, 2 mM L-Glutamine (Invitrogen), 0.1 mM Non-Essential Amino Acids (Invitrogen), 1 mM Sodium. It was cultured in DMEM (Nakaraitex) supplemented with Pyruvate (Sigma). Cells were seeded in 12-well plates and replaced with doxycycline-containing medium (0 ng / mL to 10 ng / mL) prior to transfection. Transfection was performed the day after seeding and the purified plasmid was introduced to a total volume of 400 ng. The introduction was carried out using 4 μL of Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions. The plasmids used were mixed in Opti-MEM (Invitrogen) so that the volume ratio of the switch plasmid and the trigger plasmid was 1: 1. The medium was changed 4 hours after transfection.

[Western blotting] Half of the cells to be analyzed by flow cytometry were collected in a microtube and centrifuged at 300 g, 5 min, and 4 ° C. After removing the upper sperm, 200 uL of PBS was added, resuspended, and then centrifuged at 300 g, 5 min, and 4 ° C. This process was performed twice and stored at -80 ° C. RIPA Buffer (25 mM Tris (ph7.6), 150 mM NaCl, 1% NP-40, 1% sodium deoxycholate, 0.1% SDS) was added to the collected cells at 50 uL and placed on ice for 30 min to extract the protein. This was centrifuged at 15000 g for 20 min, and the upper sperm was collected in another Eppen and stored at -20 ° C. For each sample, the protein concentration was measured using the Pierce BCA Protein Assay Kit (Thermo Scientific). The sample was then run using Extra PAGE One Precast Gel 10-20% (nacalai tesque). Markers were prepared by adding 0.5 uL MagicMark ™ XP Western Protein Standard (invitrogen) to 3 uL Full-Range Rainbow Molecular Weight Markers (GE health care). For each sample, lysate was diluted with MilliQ to a total protein concentration of 5 μg / 15 μL. After adding 6 × SDS buffer 4 uL to the prepared sample, it was boiled at 95 ° C for 3 min. The whole amount was applied to a well and electrophoresed at 20 mA for 120 min. After electrophoresis, the gel was transferred to a membrane by an iBlot drive-rotting system (Invitrogen) and blocked using Blocking One (nacalai tesque). GAPDH was detected using GAPDH Antibody (FL-335) (Santa Cruz Biotechnology) and Goat anti-Rabbit IgG (H + L) -HRP conjugate (bio-rad). LIN28A was detected using Anti-human LIN28A (Goat) (R and D Systems), Rabbit anti-Goat IgG (H + L) Secondary Antibody, and HRP conjugate (Life Technologies). After antibody treatment, chemiluminescence was performed using the ECL Plus Western Blotting Detection System (GE Healthcare) and observed with LAS-4000 (FUJIFILM).

(Example 1)
[Modification of natural aptamers]
The aptamer used in the experiment was designed by modifying the aptamer known as the LIN28A binding motif (Fig. 3, left; Original aptamer) to facilitate structure formation on mRNA. The AU base pairs present in the original sequence were gradually changed to stronger GC base pairs (Fig. 3, right; stbA, stbB, stbC). With the modification, the base pairing probability increases and the structure is expected to stabilize. CentroidFold (http://www.ncrna.org/centroidfold) was used for the secondary structure prediction of aptamers. The aptamer containing the original shown in FIG. 3 was inserted into the plasmid shown in FIG. 2 to obtain a switch plasmid. Aptamer sequences other than the stabilized aptamer sequences shown in Table 1 are shown in Table 12 below. Bases modified from the original aptamer are shown in lowercase.

[Confirmation of translation efficiency of the invented switch plasmid]
The prepared switch plasmid was co-introduced into HEK293FT cells together with a trigger plasmid expressing LIN28A. FIG. 4 is a graph comparing the translation efficiencies of each switch plasmid. The better the switch, the lower the translation efficiency. The translation efficiency was calculated by first dividing the fluorescence intensity of GFP by the fluorescence intensity of tagRFP, and then dividing the value when LIN28A was introduced and the value when LIN28A was not introduced. In addition, control switch plasmids (Fig. 4; No aptamer) were used as criteria for comparison. Error bars indicate mean ± standard deviation (n = 2, 2 independent experiments with mean taken from 3 samples each time). Stronger translational repression was observed in switches with more stabilized structures than in switches with Original inserted. However, stbD, which is considered to be the most stable in structure, did not show strong translational repression. The reason for this is that, as is clear from Fig. 3, the structure of the terminal loop has changed in stbD. Therefore, it is considered necessary to stably form an appropriate secondary structure in order to suppress translation of a stronger switch.

Next, it was confirmed whether the switch in which stbC was inserted, in which particularly strong translational repression was observed, could function in other cell types. The experiment and analysis method are the same as in the case of HEK293FT described above. FIG. 5 is a graph showing the translation efficiency of the LIN28A switch in HeLa cells. Error bars indicate mean ± standard deviation (n = 3). It was shown that the switch with stbC inserted responds to LIN28A even in HeLa cells and causes translational repression.

FIG. 6 is a graph showing the translation efficiency when the LIN28A switch or a mutant thereof is introduced into HEK293FT cells. The experiment and analysis method are the same as above. Error bars indicate mean ± standard deviation (n = 3). Mutant means a substitute of a base involved in binding to LIN28A present in the aptamer sequence. Deletion means that the base involved in the binding to LIN28A is removed from the aptamer sequence. Therefore, the binding of these two mutants to LIN28A is inhibited, and it is expected that the translation efficiency will be restored. In fact, no strong translational repression was observed in these two mutants, as was the case with stbC-inserted switches.

[Confirmation of correlation between translation efficiency of LIN28A switch and intracellular LIN28A amount]
By introducing the switch plasmid into a cell line that can induce the expression of LIN28A by adding doxycycline, the correlation between the translation efficiency of the switch and the amount of intracellular LIN28A was examined. This cell line can regulate the expression level of LIN28A depending on the concentration of doxycycline added. That is, it is expected that the fluorescence ratio of the switch decreases as the doxycycline concentration increases. FIG. 7A is a graph showing the added doxycycline concentration and the translation efficiency of each switch. It was confirmed that the switch in which stbC was inserted was most sensitively subjected to translational repression depending on the doxycycline concentration. FIG. 7B shows the results of Western blotting confirming whether the expression level of LIN28A increased in a doxycycline concentration-dependent manner. As an internal standard, GAPDH expression was also detected at the same time. In fact, it was confirmed that the expression level of LIN28A increased according to the doxycycline concentration. FIG. 7C is a graph showing the relationship between the band concentration obtained in FIG. 7B and the doxycycline concentration quantified. It was confirmed that the relative expression level of LIN28A reached the upper limit when the doxycycline concentration reached 3 to 4 ng / mL. This concentration coincides with the concentration at which the fluorescence ratio of the switch in which stbC is inserted reaches the lower limit in FIG. 7A. Therefore, it was confirmed that the LIN28A switch undergoes translational repression depending on the amount of intracellular LIN28A. FIG. 7D is a graph showing the relationship between the relative expression level and the fluorescence ratio of each switch in the section until the relative expression level of LIN28A reaches the upper limit. The fluorescence ratio of each LIN28A switch and the relative expression level of LIN28A were correlated, and it was shown that the switch with stbC inserted can detect the intracellular expression level of LIN28A with high sensitivity.

[Confirmation of translation efficiency in mRNA transfection of LIN28A switch]
We verified whether the LIN28A switch works even when protein-responsive mRNA is directly introduced into cultured cells. HEK293FT cells were co-introduced a switch mRNA, a trigger mRNA expressing LIN28A, and a reference mRNA (iRFP mRNA) to confirm that a series of mRNAs had been introduced into the cells. The amount of trigger mRNA to be introduced was changed in 6 stages of 0 ng (non-introduced), 50 ng, 100 ng, 150 ng, 200 ng, and 300 ng. Theoretically, as the amount of trigger mRNA introduced decreases, the translation efficiency from the switch mRNA decreases. FIG. 8A plots the fluorescence of the switch with stbC inserted at the time of introduction of each amount of trigger mRNA by flow cytometry. It was confirmed that the population moves downward (the amount of translation from the switch mRNA decreases) according to the amount of trigger mRNA introduced.

FIG. 8B is a dot plot in which the switch with another aptamer inserted and the measurement result of FIG. 8A are superimposed. Of the switches into which each aptamer was inserted, the movement of the cell population with the amount of trigger mRNA introduced was observed only in the switch into which stbC was inserted. Therefore, it was suggested that the cell population can be separated depending on the expression level of LIN28A in the cell by introducing a switch into which stbC is inserted into the cell population. FIG. 8C is a graph showing the translation efficiency of switch mRNA at each trigger mRNA introduction amount. The translation efficiency was calculated by first dividing the fluorescence intensity of hmAG1 by the fluorescence intensity of iRFP670, and then dividing the value when the trigger mRNA was introduced and the value when the trigger mRNA was not introduced. Of the switches with each aptamer inserted, strong translational repression was observed in the switch with stbC inserted. Furthermore, it was confirmed that the translation efficiency decreased as the amount of trigger mRNA introduced increased.

(Example 2)
[Identification of iPS cells by LIN28A switch]
LIN28A is a protein that is highly expressed in stem cells such as iPS cells and ES cells. Therefore, it is possible to distinguish between iPS cells and differentiated cells using living cells from the difference in the expression level of LIN28A. This suggests that iPS cells and differentiated cells can be distinguished from each other by the difference in translation efficiency of each cell of the LIN28A switch. In other words, when the LIN28A switch was introduced into cells, LIN28A was highly expressed in iPS cells, so translational repression should be observed, and translational repression should not be observed in differentiated cells (Fig. 9A). At this time, cells that have not been transfected with the switch mRNA can be removed by measuring the expression of the reference mRNA. Furthermore, by performing flow cytometry, the iPS cell population and the differentiated cell population can be separated from the difference in the fluorescence value of the switch mRNA with respect to the fluorescence value of the reference mRNA (Fig. 9B).

FIG. 10A is a dot plot showing the measurement results when each LIN28A switch and reference mRNA (mKO2) are co-introduced. At the control switch, the populations of iPS cells and differentiated cells matched, but it was confirmed that the cell populations were clearly separated at the switch with stbC inserted. FIG. 10B is a histogram showing the ratio of the two fluorescent signals in the dot plot of FIG. 10A. FIG. 10C is a graph comparing the translation efficiencies of the LIN28A switch in iPS cells and differentiated cells. The translation efficiency was determined by first dividing the fluorescence intensity of hmAG1 by the fluorescence intensity of mKO2, and then dividing the value in the iPS cells and the value in the differentiated cells. It was observed that the translation efficiency of the LIN28A switch in iPS cells was reduced by 50% or more compared to that in differentiated cells.

FIG. 11A is a comparison of switch translation efficiencies between iPS cells on days 14 and 34 of induction of differentiation and undifferentiated iPS cells. For translation efficiency, the fluorescence intensity of EGFP was first divided by the fluorescence intensity of tagRFP, and then the value in iPS cells and the value in differentiated cells were divided. Furthermore, the value standardized by the value of the control (No aptamer) is shown in the graph. It was confirmed that the translation efficiency of iPS cells was reduced with respect to differentiated cells. FIG. 11B is a dot plot showing the results of flow cytometry of each cell and an overlay thereof. It was observed that the cell population moved upward (the amount of translation increased) as the differentiation was induced.

(Example 3)
[Expandability of human endogenous protein detection using mRNA switch]
In this invention, we succeeded in developing a highly sensitive mRNA switch by stabilizing the structure of the aptamer inserted into mRNA. Whether or not this design principle can be applied to the detection of proteins other than LIN28A was verified by creating an mRNA switch that responds to U1A. U1A is present in human cells as a splicing-related protein. Figure 12 shows the aptamer that binds to U1A used in the experiment. U1hp and U1utr are naturally occurring aptamers. The lower secondary structure ((b) in FIG. 12) is reprinted from reference [4]. U1utr in the upper part of Fig. 12 predicts the secondary structure using Centroid Fold based on the reported sequence. As a result, U1utr was not stable and may not form the reported structure. Therefore, we designed U1utr_stb, which forms a structure similar to the reported one by modifying multiple bases. U1LSL is also an aptamer used in reference [5]. By adding a long stem structure to this aptamer, it was made difficult to interact with other regions on the mRNA, and the structure of the aptamer was protected. Aptamer sequences other than the stabilized aptamer sequences shown in Table 2 are shown in Table 13 below. Bases modified from the original aptamer are shown in lowercase.

FIG. 13 is a graph comparing the translation efficiencies of each switch plasmid. The prepared switch plasmid was co-introduced into HEK293FT cells together with a trigger plasmid expressing U1A. The translation efficiency was determined by first dividing the fluorescence intensity of GFP by the fluorescence intensity of tagRFP, and then dividing the value when U1A was introduced and the value when U1A was not introduced. In addition, control switch plasmids (Fig. 4; No aptamer) were used as criteria for comparison. Error bars indicate mean ± standard deviation (n = 3). As a result, stronger translational repression was observed in U1utr_stb, which is considered to be more stable in structure than U1utr. Furthermore, for U1LSL, stronger translational repression was observed in the aptamer in which a longer stem structure was added to stabilize the structure.

Furthermore, U1LSL and aptamers to which a stem structure was added were subjected to base substitution to prepare mutants. These mutants form the same structure as the wild type, but lose their ability to bind U1A. Therefore, it is expected that the translation efficiency will be restored in the mRNA in which the mutant is inserted. FIG. 14A is a fluorescence micrograph when each switch plasmid was introduced into HEK293FT cells together with a control trigger plasmid. Stronger translational repression was observed in switches with wild-type (WT) aptamers than in switches with mutant (mut) aptamers. FIG. 14B is a histogram obtained by flow cytometric analysis of the cells observed in FIG. 14A. In addition, FIG. 14C is a graph showing the fluorescence ratio of the switch in which the wild-type aptamer is inserted and the switch in which the mutant aptamer is inserted. The fluorescence ratio was determined by first dividing the fluorescence intensity of GFP by the fluorescence intensity of DsRed Ex, and then dividing the value of the wild type and the switch in which the mutant was inserted. Error bars indicate mean ± standard error (n = 3 independent experiments). These results suggest that the prepared U1A switch can respond to intracellular U1A. That is, by stabilizing the secondary structure of an aptamer that binds to an arbitrary protein, an mRNA switch capable of detecting a desired protein can be produced.

(Example 4) [Knockdown analysis]
The cultured cells were seeded on a 24-well plate, and the next day, a total of 500 ng of plasmid and 5 pmol of shRNA were co-introduced. The introduction was carried out using 2 μL of Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions. The plasmids used were mixed in Opti-MEM (Invitrogen) so that the volume ratio of the switch plasmid and the reference plasmid (pTAPmyc -T2A-tagRFP) was 1: 4. The medium was changed 4 to 6 hours after transfection. The day after transfection, cells were separated from the plate, passed through a mesh and analyzed by flow cytometry. In experiments with 24-well plates, Accuri C6 (BD Biosciences) was used. EGFP was detected by the FL1 (530/30 nm) filters fitter. tagRFP was detected by FL2 (585/40 nm) filters fitter. The shRNA used is as follows.

[Quantitative RT-PCR]
The day after mRNA transfection, iPS cells were separated from the plate and the cells were collected. Differentiated cells that had not been transfected were collected and used by the same method. Total RNA recovery, reverse transcription, and quantitative PCR were performed using the miRNA Cells-to-CT kit (Applied Biosystems). For the expression level of mature miRNA, miRNA TaqMan probe (Applied Biosystems) was used. In addition, the values were standardized using RNU6B. The expression level was calculated using the ΔΔCt method. The TaqMan probe used is as follows.

[Confirm that the U1A switch responds to endogenous U1A]
FIG. 15 is a graph showing the relative translation efficiency at the time of introducing shRNA in the U1A switch. The prepared switch plasmid was co-introduced into HEK293FT cells together with a reference plasmid expressing tagRFP and shRNA. The relative translation efficiency was determined by first dividing the fluorescence intensity of GFP by the fluorescence intensity of tagRFP, and then dividing the value when shRNA was not introduced and the value of each sample. Error bars indicate mean ± standard error (n = 3, mean of 3 samples each time, 3 independent experiments). As a result, an increase in translation was observed when shUA1 was introduced in the switch with a stable aptamer inserted. This result means that the intrinsic U1A can be detected with higher sensitivity in a switch in which an aptamer having a more stable structure is inserted. Therefore, by stabilizing the secondary structure, an mRNA switch having sufficient sensitivity for detecting an endogenous protein can be produced.

[Effect of introduction of LIN28A switch on intrinsic miRNA expression]
LIN28A is naturally involved in the maturation of a series of miRNAs belonging to the let7 family. Specifically, there is a LIN28A binding site at the loop site in the precursor of let7 miRNA, and LIN28A binds to this site to inhibit the expression of mature miRNA. Therefore, the introduction of an mRNA switch capable of binding to LIN28A may affect the process of controlling miRNA expression. That is, binding of the endogenous LIN28A to the mRNA switch may result in inadequate suppression of miRNA maturation and increased expression of the let7 family. To investigate this, quantitative RT-PCR was used to measure the expression levels of three typical mature let7 families.

FIG. 16 is a graph showing the expression level of mature let7 miRNA upon introduction of the LIN28A switch. The expression level in differentiated cells was expressed as 1. Error bars indicate mean ± standard deviation (n = 3). As a result, no significant change in the expression of mature miRNA was observed when the LIN28A switch was introduced into iPS cells (no aptamer, stbC). Therefore, the LIN28A switch invented this time can distinguish between iPS cells and differentiated cells without significantly affecting the expression profile of miRNA.

[Making mRNA switches using artificial aptamers]
The aptamers used in the U1A switch and LIN28A switch were created by using naturally occurring ones as they are or by using a partially extracted motif. In principle, even artificially acquired aptamers can produce highly sensitive mRNA switches by utilizing the method used this time. To verify this, an mRNA switch was prepared using an aptamer (p50A: SEQ ID NO: 83) that binds to p50, which is one of the cancer-related proteins (Fig. 17, left figure, reference [6]). By adding a long stem structure to this aptamer, it was made difficult to interact with other regions on the mRNA, and the structure of the aptamer was protected (p50A-stb: SEQ ID NO: 84, FIG. 17, right). Figure). In addition, a mutant was prepared by substituting the base of the binding site (p50A-stb _mut : SEQ ID NO: 85). FIG. 17 is an aptamer that binds to p50 used in the experiment. In preparing the mutant, the substituted bases are shown by arrows in FIG. 17 and the right figure.

FIG. 18 is a graph comparing the translation efficiencies of each switch plasmid. The prepared switch plasmid was co-introduced into HEK293FT cells together with a trigger plasmid expressing p50. The translation efficiency was calculated by first dividing the fluorescence intensity of GFP by the fluorescence intensity of tagRFP, and then dividing the value when p50 was introduced and the value when p50 was not introduced. In addition, control switch plasmids (Fig. 4; No aptamer) were used as criteria for comparison. Error bars indicate mean ± standard error (n = 3, mean of 3 samples each time, 3 independent experiments). As a result, stronger translational repression was observed in p50A-stb, which is considered to be more stable in structure than p50A. Therefore, even with artificially obtained aptamers, a highly sensitive mRNA switch can be produced by the same approach.

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Claims (4)

Method of identifying a cell endogenous protein, as the following factories:
(1) A step of introducing an intracellular protein-responsive mRNA containing an aptamer sequence specific to an intracellular protein and a marker gene sequence functionally linked thereto or a vector encoding the mRNA into a desired cell; and ( 2) Step of identifying intracellular protein based on the amount of translation of the marker gene
The aptamer sequence comprises
The method selected from the following (a) to (e):
(A) Based on the RNA secondary structure formed by the native LIN28A aptamer sequence represented by SEQ ID NO: 1, the following one or more modifications have been made to the structurally stabilized LIN28A aptamer:
(I) The AU base pairs that make up the stem containing the 5'end base and the 3'end base are replaced with GC base pairs;
(Ii) The AU base pairs that make up the stem located between the two loops are replaced with GC base pairs; or
(Iii) Base pairs 1 to 5 are added to the stem located between the two loops;
(B) Structurally stabilized LIN28A aptamer represented by any of SEQ ID NOs: 2 to 4;
(C) Structure-stabilized U1A aptamer with the following modifications based on the RNA secondary structure formed by the native U1A aptamer sequence represented by SEQ ID NO: 6.
(I) 1 to 15 AU base pairs or GC base pairs are added to the stem structure containing 5'end bases and 3'end bases, or 1 to 15 AU base pairs are added to the stem existing between the two boxes. Or GC base pairs have been added; or
Based on the RNA secondary structure formed by the U1A aptamer sequence represented by SEQ ID NO: 8, the following modified structure-stabilized U1A aptamer:
(Ii) 1 to 15 AU base pairs or GC base pairs are added to the stem containing 5'end bases and 3'end bases;
(D) Structure-stabilized p50A aptamer with the following modifications based on the RNA secondary structure formed by the p50A aptamer sequence represented by SEQ ID NO: 83:
1 to 20 AU base pairs or GC base pairs are added to the stem structure containing 5'end bases and 3'end bases;
(E) A structurally stabilized U1A aptamer represented by any of SEQ ID NOs: 7, 9, 10 and 84 . Method of identifying a cell, as the following factories:
(1) A step of introducing an intracellular protein-responsive mRNA containing an aptamer sequence specific to an intracellular protein and a marker gene sequence functionally linked to the aptamer sequence or a vector encoding the mRNA into a desired cell;
(2) A step of identifying cells expressing the protein based on the amount of translation of the marker gene.
The aptamer sequence comprises
The method selected from the following (a) to (e):
(A) Based on the RNA secondary structure formed by the native LIN28A aptamer sequence represented by SEQ ID NO: 1, the following one or more modifications have been made to the structurally stabilized LIN28A aptamer:
(I) The AU base pairs that make up the stem containing the 5'end base and the 3'end base are replaced with GC base pairs;
(Ii) The AU base pairs that make up the stem located between the two loops are replaced with GC base pairs; or
(Iii) Base pairs 1 to 5 are added to the stem located between the two loops;
(B) Structurally stabilized LIN28A aptamer represented by any of SEQ ID NOs: 2 to 4;
(C) Structure-stabilized U1A aptamer with the following modifications based on the RNA secondary structure formed by the native U1A aptamer sequence represented by SEQ ID NO: 6.
(I) 1 to 15 AU base pairs or GC base pairs are added to the stem structure containing 5'end bases and 3'end bases, or 1 to 15 AU base pairs are added to the stem existing between the two boxes. Or GC base pairs have been added; or
Based on the RNA secondary structure formed by the U1A aptamer sequence represented by SEQ ID NO: 8, the following modified structure-stabilized U1A aptamer:
(Ii) 1 to 15 AU base pairs or GC base pairs are added to the stem containing 5'end bases and 3'end bases;
(D) Structure-stabilized p50A aptamer with the following modifications based on the RNA secondary structure formed by the p50A aptamer sequence represented by SEQ ID NO: 83:
1 to 20 AU base pairs or GC base pairs are added to the stem structure containing 5'end bases and 3'end bases;
(E) A structurally stabilized U1A aptamer represented by any of SEQ ID NOs: 7, 9, 10 and 84 . The method according to claim 1 or 2, wherein the aptamer sequence is provided in the 5'UTR of the marker gene sequence. The method according to any one of claims 1 to 3, wherein the cell is a human cell.

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