WO2020183941A1 - Method for detecting microrna processing activity, and application thereof - Google Patents

Abstract

The present invention addresses the problem of providing a means for conveniently and very safely detecting microRNA processing activity. MicroRNA processing activity is detected using (1) a step of reacting, with a Drosha complex, an miRNA probe containing, in addition to the full-length sequence of a pre-miRNA, a 10-80 base length sequence connected to the 5'-end side of the pre-miRNA in a pri-miRNA and a 10-80 base length sequence connected to the 3'-end side, and containing a fluorescent substance bonded to the 5'-end region or 3'-end region thereof; and (2) a step of detecting fluorescence from a fragment that is produced by processing by the Drosha complex and contains the 5'-end region or 3'-end region of the miRNA probe.

Description

Detection method of microRNA processing activity and its application

The present invention relates to a method for detecting the processing activity of microRNA. More specifically, the present invention relates to a simple method for detecting microRNA processing activity, various assays (test methods) using the same, and the like. This application claims priority based on Japanese Patent Application No. 2019-046718 filed on March 14, 2019, and the entire contents of the patent application are incorporated by reference.

MicroRNA (miRNA) is a single-stranded RNA with a length of about 18 to 24 bases that exists in cells and becomes mature after processing in the nucleus and cytoplasm. First, it is transcribed as a primary precursor (pri-miRNA) of hundreds to thousands of bases and processed in the nucleus by the Drosha complex. The secondary precursors (pre-miRNAs) produced by this processing translocate to the cytoplasm and are further processed into mature-miRNAs by the Dicer complex. Mature miRNAs regulate the expression of target genes by binding to the target gene mRNA and inhibiting the degradation and translation of the mRNA. A single miRNA typically targets about 200 mRNAs because it binds and acts on the target binding site with incomplete sequence complementarity.

Previous studies have revealed that abnormal miRNA processing contributes to the pathophysiology of various diseases. For example, the association between DGCR8 abnormalities constituting the Drosha complex and 22q11.2 deletion syndrome (Non-Patent Document 1), and the association between MeCP2 abnormalities that interact (associate) with the Drosha complex and Rett syndrome (Non-patent). Reference 2), Relationship between TDP43 abnormality interacting with Drosha complex and amyotrophic lateral sclerosis (ALS) (Non-Patent Document 3), p53 abnormality interacting with Drosha complex and cancer Relationships (Non-Patent Document 4), associations between abnormalities in Smads interacting with the Drosha complex and cardiovascular diseases have been reported (Non-Patent Document 5).

Regarding the detection of processing activity of miRNA from pri-miRNA to pre-miRNA, a method using a radiolabel (RI) was reported in 2004 (Non-Patent Document 6). Since then, research by the same method has been carried out by several research groups (see, for example, Non-Patent Document 7).

As mentioned above, abnormal processing of miRNA is involved in the onset and progression of various diseases. If the miRNA processing activity can be easily detected and evaluated, a drug screening system using the activity as an index can be constructed, and an efficient therapeutic drug can be developed for a wide variety of diseases. It also enables objective testing or diagnosis based on evidence of genetic abnormalities. There have been reports of successful detection of miRNA processing activity. However, radiolabeling (RI) has been used to detect miRNA processing activity reported in the past (Non-Patent Document 6), and even now, it depends on the method using RI for reasons such as the problem of detection sensitivity. There is no choice but to do so (see, for example, Non-Patent Document 7). RI has problems such as high risk of itself, complicated operations and procedures, and limited facilities and places that can be used.

Therefore, the main object of the present invention is to provide a means for detecting the processing activity of miRNA easily and with high safety.

In order to solve the above problems, the present inventors considered that the site-specific use of fluorescence is advantageous not only for simplicity but also for high-sensitivity detection, and constructed a miRNA processing activity detection system using a site-specific fluorescent label. I aimed. First, the length of miRNA (miRNA probe) is a major problem in constructing this detection system. Normally, due to synthetic restrictions, the length of about 70-80 nt is the limit of synthesis by a synthesizer, and it is extremely difficult to synthesize primary-miRNA with a length of 80 nt or more even if it is short. Difficult and nearly impossible. Conventionally, an in vitro transcription reaction system has been used to prepare a primary-miRNA probe (see, for example, Non-Patent Documents 6 and 7 described above). With this method, RNA of 100 nt or more can be prepared, and it is also possible to prepare chemically labeled RNA by causing a transcription reaction using chemically labeled nucleotides as a raw material. However, it is difficult to introduce site-specific modifications by the method for preparing primary-miRNA by such an in vitro transcription reaction system. Therefore, by using the ligation reaction to synthesize and ligate 70-80nt fragments that can be synthesized by a synthesizer, a sufficiently long pri-miRNA probe that reflects the characteristic structure of pri-miRNA was prepared. , I decided to examine its effectiveness. On the other hand, we also focused on the composition of the Drosha complex used in the assay.

As a result of detailed examination, we succeeded in detecting and evaluating pri-miRNA processing activity by non-radioactive labeling by combining ligation of synthetic RNA and fluorescence detection. On the other hand, when the complex of Drosha and DGCR8 was used, the processing efficiency was significantly improved as compared with the case of Drosha alone, and a highly practical detection system was constructed. It was also shown that it is possible to evaluate the action of additional factors that control target-specific processing. Further studies have also confirmed the generality / versatility of the method (not limited to the detection of processing activity of a particular miRNA).

On the other hand, in order to apply this method, we attempted to detect the processing activity of the MeCP2 complex, which has been reported to be associated with Rett syndrome. As a result, it was possible to detect and evaluate the processing promoting action of MeCP2. This result indicates that the method is also useful for evaluating the activity of molecules (MeCP2, TDP43, p53, Smads, etc.) that interact with the Drosha complex whose main components are Drosha and DGRC8.

As described above, we have succeeded in creating an epoch-making miRNA processing activity detection system with excellent versatility and practicality. The following inventions are mainly based on the results.
[1] A method for detecting microRNA processing activity, which comprises the following steps (1) and (2):
(1) In addition to the full-length sequence of pre-miRNA, in pri-miRNA, a sequence having a length of 10 to 80 bases continuous to the 5'end side of the pre-miRNA and 10 to 80 continuous to the 3'end side. A step of reacting a Drosha complex with a miRNA probe that contains a base-length sequence and has a fluorescent substance bound to its 5'-terminal region or 3'-terminal region.
(2) A step of detecting fluorescence from a fragment containing the 5'end region or the 3'end region of the miRNA probe, which is generated by processing by the Drosha complex.
[2] The detection method according to [1], wherein the position of the fluorescent label of the miRNA probe is the 5'end or the 3'end.
[3] The detection method according to [1] or [2], wherein the length of the miRNA probe is 100 to 200 bases.
[4] The detection method according to any one of [1] to [3], wherein the miRNA probe contains a sequence of a precursor of hsa-miR-199a or a precursor of hsa-miR-214.
[5] The detection method according to any one of [1] to [4], wherein the Drosha complex contains Drosha and DGCR8.
[6] The detection method according to [5], wherein the Drosha and the DGCR8 are co-expressed in cultured cells.
[7] The detection method according to any one of [1] to [6], wherein the Drosha complex is a mutant type.
[8] The detection method according to any one of [1] to [7], wherein the reaction of step (1) is carried out in the presence of a molecule that interacts with the Drosha complex.
[9] The detection method according to [8], wherein the molecule is a transcription factor or RNA-binding protein.
[10] The detection method according to [8], wherein the molecule is MeCP2, TDP43, p53 or Smads.
[11] The detection method according to any one of [8] to [10], wherein the molecule is a mutant type.
[12] Step (1) is performed in the coexistence of the test substance.
The detection method according to any one of [1] to [11], which evaluates the action of the test substance on the processing activity based on the detection result of step (2).
[13] In addition to the full-length sequence of pre-miRNA, in pri-miRNA, a sequence having a length of 10 to 80 bases continuous on the 5'end side of the pre-miRNA and 10 to 80 continuous on the 3'end side. A reagent for detecting microRNA processing activity, which comprises a miRNA probe having a base-length sequence and whose 5'-terminal region or 3'-terminal region is fluorescently labeled.
[14] A kit for detecting microRNA processing activity, which comprises the reagent according to [13].
[15] The kit according to [14], further comprising a Drosha complex.
[16] The kit according to [15], wherein the Drosha complex comprises Drosha and DGCR8.
[17] The kit according to [15] or [16], wherein the Drosha complex is a mutant type.
[18] The kit according to any one of [15] to [17], wherein a transcription factor or RNA-binding protein that interacts with the Drosha complex is associated with the Drosha complex.
[19] The kit according to any one of [15] to [17], wherein MeCP2, TDP43, p53 or Smads are associated with the Drosha complex.

Evaluation of processing activity when Drosha alone is expressed. After the processing reaction, the generated RNA is subjected to denatured polyacrylamide gel electrophoresis (dPAGE: 10% acrylamide, 7M urea, small gel (8 cm x 8 cm), 300 V constant voltage, 20 minutes electrophoresis) for fluorescence. Was detected (left). Lane 1: Drosha (-) 45 minutes reaction, Lane 2: Drosha (+) 45 minutes reaction, Lane 3: Drosha (-) 90 minutes reaction, Lane 4: Drosha (+) 90 minutes reaction, lane 5 : Marker (XC and BPB), Lane 6: PO-61mer-Fl (AM17-CS). The processing activity was compared from the fluorescence intensity (right). Evaluation of processing activity in the case of co-expression of Drosha and DGCR8. After the processing reaction, the generated RNA is subjected to denatured polyacrylamide gel electrophoresis (dPAGE: 10% acrylamide, 7M urea, small gel (8 cm x 8 cm), 300 V constant voltage, 20 minutes electrophoresis) for fluorescence. Was detected (left). Lane 1: Control 30 minutes reaction, Lane 2: Control 60 minutes reaction, Lane 3: Drosha + DGCR8 30 minutes reaction, Lane 4: Drosha + DGCR8 60 minutes reaction. The processing activity was compared from the fluorescence intensity (center). It was also evaluated with a Primary-miR-199a probe labeled with SYBR Green II (right). Evaluation using another probe (Primary-miR-214 probe). After the processing reaction, the generated RNA is subjected to denaturing polyacrylamide gel electrophoresis (dPAGE: 10% acrylamide, 7M urea, small gel (8 cm x 8 cm), 300 V constant voltage, 35 minutes electrophoresis) for fluorescence. Was detected (left). Lane 1: Control 30 minutes reaction, Lane 2: Control 60 minutes reaction, Lane 3: Drosha + DGCR8 30 minutes reaction, Lane 4: Drosha + DGCR8 60 minutes reaction. The processing activity was compared from the fluorescence intensity (center). It was also evaluated with a Primary-miR-214 probe labeled with SYBR Green II (right). Evaluation of processing activity of MeCP2 complex. After the processing reaction, the generated RNA is subjected to denaturing polyacrylamide gel electrophoresis (dPAGE: 10% acrylamide, 7M urea, small gel (8 cm x 8 cm), 300 V constant voltage, 35 minutes electrophoresis) for fluorescence. Was detected (left). Lane 1: Control 30 minutes reaction, Lane 2: Control 60 minutes reaction, Lane 3: MeCP2 30 minutes reaction, Lane 4: MeCP2 60 minutes reaction. The processing activity was compared from the fluorescence intensity (center). It was also evaluated with a Primary-miR-214 probe labeled with SYBR Green II (right).

1. 1. MicroRNA processing activity assay 1-1. Method for Detecting MicroRNA Processing Activity The first aspect of the present invention relates to a method for detecting microRNA processing activity. As mentioned above, microRNAs (sometimes referred to as "miRNAs") become pre-miRNAs by processing with the Drosha complex and mature by further processing with the Dicer complex. As used herein, the term "microRNA processing activity (miRNA processing activity)" refers to the ability or degree (level) of the former processing, that is, processing by the Drosha complex (conversion of pri-miRNA to pre-miRNA). .. Unless otherwise stated, "processing" in the following description refers to Drosha complex-mediated processing, i.e., conversion of pri-miRNA to pre-miRNA. In the living body, the Drosha complex contains Drosha and DGCR8 as major components, and may further contain various RNA-binding proteins such as DEAD-box RNA helicase DDX5 / 17, hnRNP. It is also known that MeCP2, TDP43, p53, Smads and others associate with the Drosha complex to regulate specific miRNA processing.

In the detection method of the present invention, the following steps (1) and (2) are performed. As is clear from the configuration, the detection method of the present invention is performed in vitro. That is, it is an in vitro assay.
(1) In addition to the full-length sequence of pre-miRNA, a sequence having a length of 10 to 80 bases (preferably 20 to 50 bases) continuous with the 5'end side of the pre-miRNA and a 3'end of the pre-miRNA. The Drosha complex is reacted with a miRNA probe containing a contiguous 10 to 80 base length (preferably 20 to 50 base length) sequence on the side and fluorescently labeled with its 5'end region or 3'end region. (2) Step of detecting fluorescence from a fragment containing the 5'-terminal region or 3'-terminal region of the miRNA probe caused by processing by the Drosha complex.

Step (1)
Step (1) is a processing reaction using a miRNA probe characteristic of the present invention, in which the miRNA probe is reacted with the Drosha complex. That is, a reaction solution containing components necessary for processing, such as magnesium (Mg) and ATP, is prepared, and in the coexistence of the miRNA probe and the Drosha complex (in other words, in a state where interaction is possible), for example, 35 ° C. Incubate at ~ 39 ° C, preferably about 37 ° C. The incubation time is, for example, 10 minutes to 3 hours.

In the present invention, a miRNA probe having a characteristic sequence is prepared. Specifically, the miRNA probe is a portion of the sequence of pre-miRNA (specific precursor miRNA), which is the post-processing form, as well as the sequence of pri-miRNA, which is the pre-processing form, specifically said. A portion of pre-miRNA contiguous on the 5'end side (referred to as "pri-miRNA 5'side contiguous region" for convenience of explanation) and a portion contiguous on the 3'end side (for convenience of explanation, "pri-miRNA 3" It consists of an array containing (called'side continuous region'). Thus, the miRNA probe has a sequence that is not present in pre-miRNA but is present in pri-miRNA. The lengths of the pri-miRNA 5'side continuous region and the pri-miRNA 3'side continuous region are, for example, 10 to 80 bases long, preferably 20 to 50 bases. The inclusion of the miRNA probe in the pri-miRNA 5'side continuous region and the pri-miRNA 3'side continuous region is important for the Drosha complex to exert processing activity in the reaction of step (1). If the length of these constituent regions is too short, it will be difficult or impossible to detect the processing activity by fluorescence. On the other hand, making the pri-miRNA 5'side continuous region and the pri-miRNA 3'side continuous region longer than necessary makes it difficult to prepare the miRNA probe and is also preferable from an economic point of view (that is, preparation cost). Absent. As described below, the miRNA probe sequence is designed based on the specific miRNA sequence. Therefore, the length of the miRNA probe depends on the length of the underlying miRNA and is not uniform, but is, for example, 100 to 200 bases, preferably 130 to 160 bases. For the preparation of such a long miRNA probe, it is advisable to adopt a method that combines chemical synthesis (solid phase synthesis method, liquid phase synthesis method) and ligation reaction. Typically, the method divides the sequence of the miRNA probe of interest into two (may be divided into three or more sequences if desired), prepares each (RNA fragment) by chemical synthesis, and then RNA ligase. It is linked by a ligation reaction using. Details of the procedures / operations and conditions of the method combining chemical synthesis and ligation reaction are shown in the Examples column. When ligating RNA fragments using a chemical reaction instead of a ligation reaction, it is necessary to insert an unnatural structure at the ligation site, and the range of selection of fluorescent dyes is narrowed (for example, fluorescein). If fluorescein reacts under the conditions of a chemical reaction and the fluorescence disappears, it is desirable to use a fluorescent dye such as Cy3) when designing the configuration of the miRNA probe.

For example, the sequence of a miRNA probe can be designed based on the sequence information of various miRNAs whose existence is known. Typically, it is a sequence corresponding to the sequence of a natural miRNA, that is, a sequence consisting of a specific pre-miRNA sequence and a pri-miRNA 5'side continuous region and a pri-miRNA 3'side continuous region of the pre-miRNA. The constructed miRNA probe is used. However, a mutant miRNA probe in which a part of the sequence (for example, about 1 to 10 bases) is modified or mutated can also be used. The use of such mutant miRNA probes is useful in assessing the effect of specific mutations on specific miRNAs on processing activity.

Examples of known miRNAs that can be used in the design of miRNA probes are hsa-miR-199a and hsa-miR-214. hsa-miR-199a is a miRNA that has been reported to be associated with Rett syndrome. Therefore, when a miRNA probe corresponding to hsa-miR-199a is designed and applied to the detection method of the present invention, an assay system that can be used for research on Rett syndrome and development of a therapeutic method / drug for Rett syndrome is provided. You will be able to do it. Similarly, hsa-miR-16-1, hsa-miR-143, and hsa-miR-145 are associated with cancer, hsa-miR-21 is associated with cardiovascular disease, and hsa-miR-574 is associated with muscle. An association with atrophic lateral sclerosis (ALS) has been reported.

Research on miRNA is progressing day by day, and dramatic development is expected. With the progress of miRNA research, it is certain that many unknown miRNAs will be found, and the design of miRNA probes corresponding to such new miRNAs is naturally envisioned.

The miRNA probe is fluorescently labeled by binding a fluorescent substance to its 5'end region (typically 5'end) or 3'end region (typically 3'end). It does not prevent the fluorescent material from binding to both the 5'end region and the 3'end region, but usually binds the fluorescent material to one side.

The 5'end region and the 3'end region to be labeled sites consist of a part or all of the pri-miRNA 5'side continuous region and a part or all of the pri-miRNA 3'side continuous region, respectively, and are a Drosha complex. Included in the portion cut by processing by. Therefore, when the miRNA probe is properly processed by the Drosha complex, a fluorescently labeled fragment (a fragment containing a 5'end region or a 3'end region) will result.

The fluorescent substance used for the fluorescent label is not particularly limited. Examples of fluorescent substances are 7-AAD, Alexa Fluor® 488, Alexa Fluor® 350, Alexa Fluor® 546, Alexa Fluor® 555, Alexa Fluor®. 568, Alexa Fluor® 594, Alexa Fluor® 633, Alexa Fluor® 647, Cy ^TM 2, DsRED, EGFP, EYFP, FITC, PerCP ^TM , R-Phycoerythrin, Propidium Iodide, AMCA, DAPI, ECFP, MethylCoumarin, Allophycocyanin, Cy ^TM 3, Cy ^TM 5, SYBR® Green, Rhodamine-123, Tetramethylrhodamine, Texas Red®, PE, PE-Cy ^TM 5, PE-Cy ^TM 5.5, PE-Cy ^TM 7, APC, APC-Cy ^TM 7, Oregon Green, Carboxyfluorescein, Carboxyfluorescein diacetate, Pyrene, HyLite, Quantum Dot. Fluorescent labeling can be performed by a conventional method, and for example, a method such as automatic nucleic acid synthesis using a phosphoramidite form of a fluorescent molecule (for example, 6-Fluorescein Phosphoramidite manufactured by Glen Research) may be adopted. The miRNA probe may be labeled using a commercially available RNA fluorescent labeling kit (for example, EndTag Nucleic Acid Labeling System manufactured by Vector Laboratories).

Two or more types of miRNA probes that are fluorescently labeled so as to be distinguishable from each other may be prepared and subjected to the reaction of step (1). This embodiment provides a detection system capable of simultaneously evaluating the processing activity of two or more types of miRNA probes. In this embodiment, fluorescence derived from each miRNA probe is detected in step (2) described later.

The Drosha complex used in step (1) contains Drosha (drosha ribonuclease III) as an essential component. Drosha is a protein with a molecular weight of approximately 160 kDa and contains two RNase III domains. There are DGCR8 binding sites on each of these domains. Drosha forms a complex (called the Drosha complex or Drosha-DGCR8 complex) with DGCR8 and processes pri-miRNA. In consideration of the fact that the processing efficiency was significantly improved when Drosha and DGCR8 were used in combination in addition to the structure of the Drosha complex in a living body (see Examples described later), the Drosha complex containing Drosha and DGCR8 is preferably the present invention. Is used as a detection method for.

Drosha is widely preserved across species. In the present invention, human Drosha is typically used, but Drosha of other species (for example, mouse, rat, Drosophila, etc.) may be adopted depending on the purpose. In that case, as for other components (for example, DGCR8) constituting the Drosha complex, those of the same kind are used in principle. Human Drosha is registered in the NCBI (National Center for Biotechnology Information) database with Gene ID: 29102 (drosha ribonuclease III [Homo sapiens (human)]). The amino acid sequence of human Drosha isoform 1 is set to SEQ ID NO: 1 (DEFINITION: ribonuclease 3 isoform 1 [Homo sapiens]. ACCESSION: NP_037367), and the amino acid sequence of isoform 2 is set to SEQ ID NO: 2 (DEFINITION; ribonuclease 3 isoform 2 [Homo]. sapiens]. ACCESSION: NP_001093882).

DGCR8 binds to Drosha to form a Drosha complex. DGCR8 has an RNA-binding domain that assists Drosha in processing pri-miRNA by binding to pri-miRNA. Human DGCR8 is registered in the NCBI (National Center for Biotechnology Information) database with Gene ID: 54487 (DGCR8, microprocessor complex subsystem [Homo sapiens (human)]). The amino acid sequence of human DGCR8 (isoform 1) is shown in SEQ ID NO: 3 (DEFINITION: microprocessor complex subunit DGCR8 isoform 1 [Homo sapiens]. ACCESSION: NP_073557 NP_073612).

The Drosha complex used in step (1) is obtained, for example, by recovering Drosha forcibly expressed in cultured cells (for example, HEK293 cells, CHO cells, COS cells) that can be used for expression / preparation of foreign proteins. Can be prepared (purified as needed). If it is a Drosha complex containing DGCR8 in addition to Drosha, the same method is used, that is, by recovery from cultured cells co-expressing Drosha and DGCR8, or after Drosha and DGCR8 are prepared separately, both are mixed and combined. It can be prepared by embodying. Instead of preparing the Drosha complex as a recombinant protein, the Drosha complex can also be prepared by separating and purifying the cells separated from the living body or its subculture cells.

The Drosha complex used in the present invention may contain elements / factors other than Drosha and DGCR8. Examples of elements / factors here are various RNA-binding proteins such as DEAD-box RNA helicase DDX5 / 17, hnRNP, which are constituent factors of the Drosha complex in the living body.

The Drosha complex may be a mutant type. When the mutant Drosha complex is used, it becomes an assay system useful for research on diseases related to processing abnormalities and development of therapeutic agents (see the column 1-2. For details). A mutant Drosha complex is a Drosha complex in which at least one of its constituents contains a mutation (which is not a normal structure). For example, in the case of a Drosha complex containing Drosha and DGCR8, Drosha or DGCR8, or those containing mutations in both of them, correspond to the mutant Drosha complex. It may be either a naturally occurring mutation or an artificially occurring mutation. As the former mutation, for example, a mutation found in the Drosha complex in a patient cell exhibiting a processing abnormality is assumed. On the other hand, when preparing a Drosha complex as a recombinant protein, by designing the expression cost lacto so as to include the intended mutation, a mutant Drosha complex containing a mutation by an artificial operation can be obtained.

In one aspect, step (1) is performed in the presence of a molecule that interacts with the Drosha complex (hereinafter referred to as "interacting molecule"). For example, molecules that associate with the Drosha complex and are involved in the processing of specific miRNAs (specific examples are various transcription factors and RNA-binding proteins such as MeCP2, TDP43, p53, and Smads) are interacting molecules. If such an interacting molecule is used, for example, the molecule may be added to the reaction system (specifically, added to the reaction solution) in step (1). Alternatively, a Drosha complex in which the molecules are associated, that is, a Drosha complex containing an interacting molecule may be prepared and subjected to the reaction. The Drosha complex containing the interacting molecule is the same method as when preparing the Drosha complex containing Drosha and DGCR8, i.e., a culture in which the interacting molecule is forcibly expressed together with Drosha and other necessary molecules (eg, DGCR8). It can be prepared by collecting from cells or by using a method such as immunoprecipitation targeting an interacting molecule to separate and purify cells separated from cultured cells or living organisms or subcultured cells thereof. Human MeCP2 is registered in the NCBI (National Center for Biotechnology Information) database with Gene ID: 4204 (methyl-CpG binding protein 2 [Homo sapiens (human)]). The amino acid sequence of isoform 1 of human MeCP2 is set to SEQ ID NO: 4 (DEFINITION: methyl-CpG-binding protein 2 isoform 1 [Homo sapiens]. ACCESSION: NP_004983), and the amino acid sequence of isoform 2 is set to SEQ ID NO: 5 (DEFINITION: methyl). -CpG-binding protein 2 isoform 2 [Homo sapiens]. ACCESSION: NP_001104262), respectively.

Mutants may also be used for the interacting molecule. As in the case of the above-mentioned mutant Drosha complex, mutations in interacting molecules are roughly classified into naturally occurring ones and artificially occurring ones. If a mutant interacting molecule is used, it becomes a useful assay system for research on diseases related to processing abnormalities caused by the interacting molecule and development of therapeutic agents (see the column 1-2. For details). ..

Step (2)
After the reaction in step (1), fluorescence is detected (step (2)). Specifically, fluorescence from a fragment (fluorescent-labeled fragment) containing the 5'-terminal region or 3'-terminal region of the miRNA probe generated by processing by the Drosha complex is detected. Successful processing of miRNA probes results in fragments containing the binding site of the fluorescent material (5'end or 3'end region of the miRNA probe). Fluorescence derived from the fragment thus produced will be detected.

For example, after the reaction in step (1), the reaction product is separated by electrophoresis or the like, and fluorescence is detected. When electrophoresis is used, a fluorescently labeled fragment is observed as a band that emits fluorescence. According to the present invention, since a miRNA probe that is fluorescently labeled specifically at the cleavage site by processing is used, the portion cut by processing (fragment having a small molecular weight) exhibits fluorescence and has high sensitivity (high S / N ratio). ) Can be detected. In contrast, in the conventional method using RI labeling, the miRNA probe labeled specifically for the cleavage site cannot be used due to the characteristics of RI labeling, and signals from other than the portion cut by processing can be detected. As a result, the S / N ratio decreases.

Various fluorescence detectors / fluorescence measuring devices can be used to detect fluorescence. Examples of fluorescence detectors / fluorescence measuring devices are Biorad's ChemiDoc ^TM XRS + system and Horiba's modular fluorescence spectrophotometer Fluorolog-3. The detection result can be quantified using dedicated or general-purpose software (for example, Image Lab ^TM software of Biorad, FluoroEssence V3 of Horiba) or the like. The detected value and the quantified data reflect the processing activity. The processing activity can be evaluated by analyzing the data.

1-2. Evaluation system using microRNA processing activity In one aspect of the detection method of the present invention, step (1) is performed in the coexistence of a test substance, and based on the detection result of step (2), the test substance for processing activity Evaluate the action of. This aspect serves as an evaluation system for drug efficacy, toxicity, etc. targeting the mi-RNA processing mechanism. For example, by using a mutant Drosha complex that causes processing abnormalities, the effect (effect) of the test substance on the mutant Drosha complex can be evaluated, and processing abnormalities caused by the mutant Drosha complex can cause onset or progression. It is useful for researching various diseases, developing therapeutic agents, and developing diagnostic methods. In addition, if a mutant interaction molecule is used, it becomes a useful evaluation system for research on diseases in which processing abnormalities caused by the interaction molecule cause the onset and progression, development of therapeutic agents, development of diagnostic methods, and the like. Since processing abnormalities caused by interacting molecules are highly disease-specific, the evaluation system is extremely useful as a research tool targeting a specific disease or disease group. The mutant Drosha complex and the mutant interacting molecule may be used in combination.

On the other hand, when the normal Drosha complex is used, it can be used and applied to, for example, toxicity evaluation of existing drugs or new drugs. The term "toxicity" should be interpreted in a broad sense, and in addition to general toxicity (acute toxicity, subacute toxicity, chronic toxicity), side effects, carcinogenicity, mutagenicity, teratogenicity, etc. are also one of the toxicity. ..

If the test substance is found to have an effect of correcting or ameliorating processing abnormalities in the detection method using the mutant Drosha complex and / or the mutant interacting molecule, the substance is a new drug (pharmaceutical) or its lead compound. It is promising as such. As described above, the detection method of this embodiment is particularly useful as a drug efficacy evaluation system or a drug screening system. An example of an embodiment in which the present invention is applied to the evaluation of the efficacy of a test substance will be described. First, from patient-derived cells, for example, extracts of affected site cells (for example, cancer cells) and iPS cells (disease iPS cells), less invasive skin-derived cells (fibroblasts, etc.), or these cells. A sample consisting of the recovered Drosha complex (which may be an association of specific interacting molecules) is prepared and reacted with a specific miRNA probe that is relevant to the disease affecting the patient. At the time of the reaction, the test substance is added to the reaction solution. Alternatively, the sample is treated with the test substance prior to the reaction. After the reaction, fluorescence is detected and compared to the control (typically the detection result in the absence of the test substance). If the detected value is significantly higher than that of the control, it can be judged that the test substance has an effect of correcting or ameliorating the processing abnormality. A test substance having such efficacy is promising as an active ingredient or a candidate for a therapeutic agent for a disease affecting a patient.

Typically, in this embodiment, step (1) is performed in the presence or absence of the test substance, and the detection result of step (2) in the former case and the detection result of step (2) in the latter case are obtained. Compare. If the effect of the test substance on the processing activity is evaluated using the comparison with the control (control) in this way, highly objective and reliable evaluation results can be obtained.

The test substance is not particularly limited. Various substances that need to be evaluated for their efficacy or toxicity can be test substances. Organic compounds or inorganic compounds of various molecular sizes can be used as the test substance. Examples of organic compounds are nucleic acids, peptides, proteins, lipids (simple lipids, complex lipids (phosphoglycerides, sphingolipids, glycoglycerides, cerebrosides, etc.), prostaglandins, isoprenoids, terpenes, steroids, polyphenols, catechins, vitamins (B1,) B2, B3, B5, B6, B7, B9, B12, C, A, D, E, etc.) can be exemplified. Existing or candidate ingredients such as pharmaceuticals, nutritional foods, food additives, pesticides, cosmetics (cosmetics), etc. Is also one of the preferred test substances. A plant extract, a cell extract, a culture supernatant, or the like may be used as a test substance. The action, synergistic action, etc. may be investigated. The test substance may be derived from a natural product or may be synthesized. In the latter case, for example, a method of combinatorial synthesis is used for efficiency. It is possible to construct a simple assay system.

2. 2. Reagents and Kits for Detecting Processing Activity As a second aspect of the present invention, reagents (reagents for detecting miRNA processing activity) and kits (kits for detecting miRNA processing activity) that enable the detection method of the present invention to be carried out more easily. )I will provide a. Hereinafter, the configurations, features, and the like of the reagents and kits of the present invention will be described, but matters not mentioned are as described in the first aspect, and the corresponding explanations are incorporated.

The reagent of the present invention consists of a miRNA probe, and in addition to the full-length sequence of a specific pre-miRNA, the pri-miRNA has a length of 10 to 80 bases (preferably 20 to 50) continuous to the 5'end side of the pre-miRNA. Base length) sequence (pri-miRNA 5'side continuous region) and 10 to 80 base length (preferably 20 to 50 base length) sequence (pri-miRNA 3'side continuous region) continuous to the 3'end side ), And its 5'end region or 3'end region is fluorescently labeled. In the kit of the present invention, the reagent is contained as an essential component. If two or more reagents designed based on different miRNA sequences are used as components of the kit, the kit can detect the processing activity for the processing of two or more miRNAs.

In a preferred embodiment, the Drosha complex is also a component of the kit. In the case of the kit of this embodiment, the processing activity can be detected without separately preparing the Drosha complex. Preferably, a Drosha complex containing Drosha and DGCR8 is adopted to obtain a kit with high processing efficiency. In addition to DGCR8, other constituent factors (for example, various RNA-binding proteins such as DEAD-box RNA helicase DDX5 / 17, hnRNP, etc.) may be included in the Drosha complex. In addition, if a mutant Drosha complex is adopted, it will be a useful kit for, for example, research on diseases related to processing abnormalities and development of therapeutic agents.

As the Drosha complex, we adopted a complex of interacting molecules such as MeCP2, TDP43, p53, and Smads, and researched diseases in which processing abnormalities caused by the interacting molecules cause the onset and progression, and development of therapeutic agents. The kit may be suitable for the development of a diagnostic method. A mutant interaction molecule may be adopted as a kit particularly useful in research on diseases related to processing abnormalities caused by the interaction molecule, development of therapeutic agents, and the like.

As a control, the kit includes a miRNA probe designed based on a sequence of miRNA that generally has constitutive expression, and a miRNA probe designed based on a sequence of miRNA that has constitutive expression in a specific cell or tissue. It is good.

Other elements that can be included in the kit include solutions for processing reactions (including, for example, magnesium and ATP), buffers (for reaction, dilution, washing, etc.), and reaction vessels. In addition, an instruction manual is usually attached to the kit of the present invention.

Processing of miRNA is important for various biological phenomena, and its abnormalities are involved in the onset and progression of brain diseases, cardiovascular diseases, cancer, etc. The following studies were conducted with the aim of creating a new means for safely and easily detecting miRNA processing activity, which is clinically important as well as a research subject.

1. 1. Evaluation of processing activity by fluorescently labeled miRNA probe (1)
1-1. Method (1) Preparation of Primary-miR-199a probe (1-1) Chemosynthesis of nucleic acid fragment Two RNA oligonucleotides required for preparation of fluorescently labeled miRNA probe "Primary-miR-199a probe" corresponding to miR-199a (SEQ ID NO: 6 and SEQ ID NO: 7) were prepared by the following methods. The Primary-miR-199a probe is a 140 mer sequence (SEQ ID NO: 9) in which a sequence derived from pri-miR-199a is added to both ends of the precursor (pre-miR-199a) (SEQ ID NO: 8) of miR-199a. ), And its 3'end is labeled with fluorescein.

For the preparation of RNA oligonucleotides, 2'-TOM (triisopropylsilyloxymethyl) protected β-cyanoethyl phosphoramidite (DMT-2'-O-TOM-rA (Ac), DMT-2'-O-TOM- rG (Ac), DMT-2'-O-TOM-rC (Ac), DMT-2'-O-TOM-rU) (Glen Research or ChemGenes) were used. Each phosphoromidite monomer is prepared so as to be a 0.05 mol / L acetonitrile solution, and synthesized by a DNA / RNA solid phase synthesizer (NTS M-2-MX, Nippon Techno Service Co., Ltd.) using 0.8 μmol of a solid phase carrier. did. Universal UnyLinker Support 2000Å (ChemGenes) was used as the solid phase carrier, and the condensation time for the first base was 15 minutes, and the subsequent condensation time was 3 minutes each. Phosphorylation of the hydroxyl group at the 5'end was performed using 5'-Phosphate-ON Reagent (0.05 mol / L acetonitrile solution, ChemGenes). DMT-6-FAM phosphoramidite (ChemGenes) was used as the fluorescein label.

The reagents used in the solid phase synthesizer are as follows. To remove the dimethoxytrityl group of the hydroxyl group at the 5'end, a reaction was carried out for 10 seconds using a commercially available deblocking reagent (Deblocking Solution-1, 3 w / v% trichloroacetic acid / dichloromethane solution, Wako Pure Chemical Industries, Ltd.). It was. A commercially available activator solution (activator solution 3, Wako Junyaku Co., Ltd.) was used as an activator for phosphoramidite. A commercially available capping solution (Cap A solution-2 and Cap B solution-2, Wako Pure Chemical Industries, Ltd.) was used for capping the unreacted 5'-terminal hydroxyl group, and the reaction was carried out for 10 seconds. As an oxidizing agent for producing a phosphoric acid ester, a solution containing pyridine, THF, water and iodine (Oxidizer, 0.01 M iodine, 29.2% water, 6.3% pyridine, 64.5% acetonitrile, Honeywell) was used, and 10 Reacted for seconds. After solid-phase synthesis, the dimethoxytrityl group of the hydroxyl group at the 5'end of the RNA oligonucleotide was deprotected on the solid-phase carrier. The synthesized RNA oligonucleotide is basically deresinized under the following conditions, that is, "treatment with concentrated aqueous ammonia: 40% methylamine aqueous solution = 1: 1 (Wako Pure Chemical Industries, Ltd.) at 65 ° C. for 1 hour".・ Deprotected. The fluorescein-labeled RNA was treated with concentrated aqueous ammonia for 30 minutes at room temperature, an equal amount of 40% aqueous methylamine solution was added, and the RNA was treated at 65 ° C. for 1 hour. After completely drying the solution obtained in the resin removal process by concentration with a centrifugal evaporator, a 2'hydroxyl TOM protecting group was used using tetrabutylammonium fluoride (1M tetrahydrofuran solution, Tokyo Chemical Industry Co., Ltd., 1 mL). (Treatment at 50 ° C for 10 minutes followed by treatment at 35 ° C for 6 hours). Tris-HCl buffer (Tris-HCl, 1M, pH 7.4, 1 mL) was added to the solution and mixed, and then tetrahydrofuran was removed by concentration with a centrifugal evaporator. The obtained solution was applied to a gel filtration column (NAP-25, GE Healthcare) equilibrated with ultra-pure water and treated by a product protocol. Fractions containing RNA oligonucleotides were concentrated by a centrifugal evaporator and then purified using a modified polyacrylamide gel (hereinafter, dPAGE).

(1-2) Purification of RNA fragments using dPAGE An aqueous solution of ammonium persulfate (hereinafter referred to as APS) and N, N, N', N'-in an acrylamide gel solution (including 7M urea as a denaturant) at each% concentration. A gel was prepared by adding tetramethylenediamine (hereinafter, TEMED) as a polymerization agent and allowing it to solidify (room temperature, 6 to 12 hours). RNA samples were mixed with gel loading buffer (80% formamide, TBE) and loaded onto gels. After electrophoresis, RNA bands were detected by UV irradiation (254 nm) and cut out of the gel using a razor blade. After finely crushing the cut gel pieces, RNA was extracted with ultrapure water (shaking at room temperature for 12 to 24 hours). The RNA extract was desalted and concentrated using Amicon Ultra 10K (Millipore), and ethanol precipitation (0.3M sodium acetate (pH 5.2) / 70% ethanol) was performed to obtain RNA pellets. RNA pellets were rinsed with 80% ethanol and then dried on a centrifugal evaporator. The obtained RNA pellet was dissolved in ultrapure water and diluted to an appropriate concentration. The absorbance (260 nm) of each diluent was measured by ultraviolet-visible absorptiometry (NanoDrop, Thermo scientific), and the concentration of each RNA oligonucleotide was determined from the molar extinction coefficient of each RNA sequence (molar extinction coefficient of each RNA sequence). Was calculated using a web-based program from Integrated DNA Technologies).

(1-3) Synthesis of probe by enzyme ligation reaction RNA oligonucleotide (SEQ ID NO: 6) and RNA oligonucleotide (SEQ ID NO: 7) (each final concentration 1 μM) are added to a T4 RNA ligase buffer solution (final concentration 50 mM Tris-). A sample dissolved in HCl (pH 7.5), 10 mM MgCl ₂ , 10 mM DTT, 1 mM ATP, Takara Bio Co., Ltd.) was heated at 90 ° C. for 5 minutes and then slowly cooled to room temperature. 60% PEG6000 was added to this solution (final concentration 15%), followed by T4 RNA ligase (Takara Bio, final concentration 0.2 unit / μL), mixed, and then allowed to stand at room temperature for 16 hours. The same volume of TE saturated phenol solution (Nacalai Tesque, Inc.) was added to the reaction solution, and the mixture was centrifuged, and the upper layer was separated into another tube. Chloroform was added to this solution, mixed with a vortex mixer, centrifuged, and the upper layer was recovered to obtain RNA pellets by alcohol precipitation (0.3 M aqueous sodium acetate solution (pH 5.2) / 70% ethanol). This RNA was separated by gel electrophoresis using a 6% denatured polyacrylamide gel. The target band was excised and gel extracted to obtain a Primary-miR-199a probe (the purification operation was the same as for the purification of the RNA oligonucleotide described above).

(2) Primary-miRNA processing assay Five HEK293T cells (Invitrogen) were prepared in a 15 cm dish in a 70-80% confluent state. To 3 ml of OPTI-MEM (Invitrogen) (amount per 15 cm dish), 60 μl of PEI (Polyethylenimine; polyscience) 1 mg / ml and 15 μg of plasmid DNA (CSII-FLAG-Drosha 15 μg) were added in this order and incubated for 15 to 20 minutes. Later, the cells were added to the culturing dish. The next day, the medium was changed and the culture was continued. After 1 day, cells were washed once with ice-cold PBS and ice-cold Lysis buffer (0.5% NP40, 150 mM NaCl, 10 mM Tris-HCl [pH 7.4]) was added to the dish. Cell extracts were collected and incubated on ice for at least 10 minutes. Then, the cell extract was sonicated (10 seconds) three times on ice. After centrifugation at 15,000 rpm at 4 ° C. for 15 minutes, the soluble fraction was collected, anti-FLAG antibody-conjugated beads were added, and the mixture was shaken at 4 ° C. for 2 to 3 hours for reaction. The supernatant was removed by centrifugation at 4,000 rpm and 4 ° C. for 5 minutes. Lysis buffer was added, mixed by inversion, and then centrifuged. This operation was repeated and the beads were washed 4 times. Processing buffer (20 mM Tris-HCl [pH 7.9], 0.1 M KCl, 10% Glycerol, 5 mM dithiothreitol [DTT], 0.2 mM PMSF) was added, and the same washing was performed once. Remove the supernatant and add 3 μl of Energy solution [64 mM MgCl ₂ , 10 mM ATP, 200 mM Creatine phosphate] to 23 μl of Processing buffer, 1.5 μl of RNase inhibitor, and 30 μg of Creatine kinase to make the final 30 μl. 0.5 μl of / ml and 10 pmol of Primary-miR-199a probe) were added to 30 μl beads, and a processing assay was performed at 37 ° C. After 45 minutes and 90 minutes, 100 μl of TE-saturated phenol and 100 μl of WQ water were added, and the reaction was terminated by vigorous mixing. The supernatant was centrifuged at 14,000 rpm and 4 ° C. for 5 minutes, and the aqueous layer of the supernatant was collected. RNA was purified from the recovered aqueous layer and then electrophoresed (dPAGE) to detect fluorescence.

1-2. Results / Discussion Even with Drosha alone expression, a slight increase in cleaved fragments over time was confirmed (Fig. 1). Processing activity by the Drosha complex could be detected, but the activity level was very low and it was difficult to use in the evaluation system.

2. 2. Evaluation of processing activity by fluorescently labeled miRNA probe (2)
2-1. Method 1-1. Using the Primary-miR-199a probe prepared by the above method, the processing activity when Drosha and DGCR8 were co-expressed was evaluated. The processing assay and fluorescence detection were in accordance with the above experiment (1-1.). However, in order to co-express Drosha and DGCR8, 30 μg of plasmid DNA (CSII-FLAG-Drosha 15 μg, GFP-DGCR8 15 μg) was used for transfection of HEK293T cells. The reaction time of the processing assay was 30 minutes and 90 minutes.

2-2. Results / Discussion The miRNA processing efficiency was greatly improved, and fluorescence evaluation using a photometer was also possible (Fig. 2). It was shown that the use of the complex of Drosha and DGCR8 significantly improved the processing efficiency and made the detection system highly practical. By using this experimental system, it can be applied to drug screening systems and diagnostic methods.

3. 3. Evaluation of processing activity by fluorescently labeled miRNA probe (3)
3-1. Method In order to verify the generality / versatility of the detection system using the complex of Drosha and DGCR8, the processing activity was evaluated with another probe "Primary-miR-214 probe". The Primary-miR-214 probe promotes the formation of tree processes and is involved in neural function miR-214 (Irie K., Tsujimura K. et al, J Biol Chem. 2016 Jun 24; 291 (26): 13891 -904) corresponds to. The Primary-miR-214 probe consists of a 160 mer sequence (SEQ ID NO: 11) with a sequence derived from pri- miR-214 added to both ends of the miR-214 precursor (pre-miR-214) (SEQ ID NO: 10). .. Its 3'end was labeled with fluorescein or SYBR Green II. The adjustment method of the Primary-miR-214 probe is 1-1. It is the same as (1). The processing assay and fluorescence detection were in accordance with the above experiment (2-1.). However, the reaction time of the processing assay was 30 minutes and 60 minutes.

3-2. Results / Discussion Fluorescence was able to detect cleavage of the Primary-miR-214 probe with high sensitivity (Fig. 3, left, center). It was confirmed that this method can be widely applied to the detection of processing activity of primary-miRNA, that is, the generality / versatility of this method. This result suggests that this method can be applied to primary-miRNA in general. In addition, by selling it as a kit, many researchers will be able to evaluate the processing of primary-miRNA, and it is expected that miRNA will contribute to the related advances in basic biology and medicine. Under these conditions, even when the probe was fluorescently labeled with SYBR Green II instead of fluorescein, processing activity was detected with a certain degree of sensitivity (Fig. 3, right).

4. Evaluation of processing activity of MeCP2 complex 4-1. Method In order to further verify the usefulness of this method and to apply it, the processing activity of the MeCP2 complex was evaluated. Fluorescein-labeled Primary-miR-199a probe and SYBR Green II-labeled Primary-miR-199a probe were used as probes. The processing assay and fluorescence detection were in accordance with the above experiment (2-1.). However, in order to express the MeCP2 complex, HEK293 cells were conditioned to co-express MeCP2 and DDX5 in addition to Drosha and DGCR8. The reaction time of the processing assay was 30 minutes and 60 minutes.

4-2. Results / Discussion The miRNA processing activity of the MeCP2 immunoprecipitate could be detected with high sensitivity by fluorescence, and the primary-miRNA processing promoting action of MeCP2 was successfully reproduced (Fig. 4, left, center). From this result, it was clarified that the processing activity of the interacting molecule can be evaluated by this method. In other words, it suggests that it can be applied to the diagnosis of diseases caused by dysfunction of interacting molecules such as MeCP2, p53 and Smad, and the development of therapeutic agents. When the probe was fluorescently labeled with SYBR Green II instead of fluorescein, processing activity was detected with a certain degree of sensitivity (Fig. 4, right).

We have succeeded in establishing a detection system that can evaluate miRNA processing activity by fluorescence, which is a non-radioactive label. Since the detection system of the present invention does not use a radiolabel (RI), it is possible to safely and easily detect miRNA processing activity, which contributes to the progress and popularization of highly difficult miRNA research. For example, as a tool for pathological research or basic research on brain diseases (for example, Let syndrome, 22q11.2 deletion syndrome, ALS), cardiovascular diseases, cancer, etc., an evaluation system effective for developing therapeutic agents for these diseases (for example) For example, it can be expected that the present invention will be used as a means for testing / diagnosing these diseases as (for example, drug screening).

Utilization and application of the present invention will lead to new discoveries and understanding of biological phenomena through miRNA processing. For example, there is a possibility that a new disease caused by an abnormality or disorder in miRNA processing may be found, and it can be expected to be applied to the diagnosis of a disease whose etiology is unknown.

The present invention is not limited to the description of the embodiments and examples of the above invention. Various modifications are also included in the invention without departing from the scope of the claims and within the scope that can be easily conceived by those skilled in the art. The contents of the papers, published patent gazettes, patent gazettes, etc. specified in this specification shall be cited by reference in their entirety.

SEQ ID NO: 6: Description of artificial sequence: RNA oligonucleotide SEQ ID NO: 7: Description of artificial sequence: RNA oligonucleotide SEQ ID NO: 8: Description of artificial sequence: pre-miR-199a
SEQ ID NO: 9: Description of artificial sequence: Primary-miR-199a probe SEQ ID NO: 10: Description of artificial sequence: pre-miR-214
SEQ ID NO: 11: Description of artificial sequence: Primary-miR-214 probe

Claims (19)

1. Methods for detecting microRNA processing activity, including the following steps (1) and (2):
   (1) In addition to the full-length sequence of pre-miRNA, in pri-miRNA, a sequence having a length of 10 to 80 bases continuous to the 5'end side of the pre-miRNA and 10 to 80 continuous to the 3'end side. A step of reacting a Drosha complex with a miRNA probe that contains a base-length sequence and has a fluorescent substance bound to its 5'-terminal region or 3'-terminal region.
   (2) A step of detecting fluorescence from a fragment containing the 5'end region or the 3'end region of the miRNA probe, which is generated by processing by the Drosha complex.
2. The detection method according to claim 1, wherein the position of the fluorescent label of the miRNA probe is at the 5'end or the 3'end.
3. The detection method according to claim 1 or 2, wherein the length of the miRNA probe is 100 to 200 bases.
4. The detection method according to any one of claims 1 to 3, wherein the miRNA probe contains a sequence of a precursor of hsa-miR-199a or a precursor of hsa-miR-214.
5. The detection method according to any one of claims 1 to 4, wherein the Drosha complex contains Drosha and DGCR8.
6. The detection method according to claim 5, wherein the Drosha and the DGCR8 are co-expressed in cultured cells.
7. The detection method according to any one of claims 1 to 6, wherein the Drosha complex is a mutant type.
8. The detection method according to any one of claims 1 to 7, wherein the reaction of step (1) is carried out in the presence of a molecule that interacts with the Drosha complex.
9. The detection method according to claim 8, wherein the molecule is a transcription factor or an RNA-binding protein.
10. The detection method according to claim 8, wherein the molecule is MeCP2, TDP43, p53 or Smads.
11. The detection method according to any one of claims 8 to 10, wherein the molecule is a mutant type.
12. Step (1) is performed in the presence of the test substance.
    The detection method according to any one of claims 1 to 11, which evaluates the action of the test substance on the processing activity based on the detection result of step (2).
13. In addition to the full-length sequence of the pre-miRNA, the pri-miRNA has a sequence having a length of 10 to 80 bases continuous to the 5'end side of the pre-miRNA and a sequence having a length of 10 to 80 bases continuous to the 3'end side. A reagent for detecting microRNA processing activity, which comprises a miRNA probe having a sequence and whose 5'end region or 3'end region is fluorescently labeled.
14. A kit for detecting microRNA processing activity, which comprises the reagent according to claim 13.
15. The kit according to claim 14, further comprising a Drosha complex.
16. The kit according to claim 15, wherein the Drosha complex comprises Drosha and DGCR8.
17. The kit according to claim 15 or 16, wherein the Drosha complex is a mutant type.
18. The kit according to any one of claims 15 to 17, wherein a transcription factor or RNA-binding protein that interacts with the Drosha complex is associated with the Drosha complex.
19. The kit according to any one of claims 15 to 17, wherein MeCP2, TDP43, p53 or Smads are associated with the Drosha complex.

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