JP2010104297A - METHOD FOR DETECTING RNA-CLEAVING REACTION BY siRNA - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for easily and quickly detecting RNA-cleaving reaction with siRNA used in RNA interference by applying a single molecule fluorometry technique. <P>SOLUTION: The method for detecting the RNA-cleaving reaction with siRNA used in RNA interference is characterized by use of a fluorescent labeled single-strand RNA as a substrate RNA for the RNA-cleaving reaction and detection of whether the substrate RNA is cleaved by siRNA or not by the single molecule fluorometry. There are also provided the above detection method wherein the substrate RNA is a single-strand RNA labeled with a fluorescent material at 3' terminal and 5' terminal, either one of the above detection methods wherein the single molecule fluorometry is fluorescence intensity multiple distribution analysis, and either one of the above detection methods wherein the RNA-cleaving reaction with siRNA is carried out in a cell or in a reaction solution. <P>COPYRIGHT: (C)2010,JPO&INPIT

Description

  The present invention relates to a method for detecting RNA cleavage reaction by siRNA used in RNA interference by single molecule fluorescence analysis.

  The RNA interference method was reported by Fire et al. In 1998 as a phenomenon that causes gene-specific suppression when double-stranded RNA is introduced into the body of a nematode (see, for example, Non-Patent Document 1 and Patent Document 1). .) Later, it became clear that this phenomenon exists not only in nematodes but also in a wide range of species such as flies, plants and animals. However, in mammals such as humans, it has also been known that interferon responses are caused by introducing double-stranded RNA of 30 base pairs or more (see, for example, Non-Patent Documents 2 and 3). The interferon response is a defense mechanism of the living body that is known to work in advance on the immune response, and is known to cause nonspecific degradation of single-stranded RNA. Subsequently, Elbashir et al. Have shown that by introducing 21 base pair double stranded RNA (siRNA) into cells, the interferon response is avoided and sequence-specific RNA interference occurs (eg, Non-patent document 4). This experiment has opened the way to apply RNA interference technology even in mammals.

  As a result of success in mammals, the expectation that RNA interference technology could be applied as a nucleic acid drug has grown. Nucleic acid drugs have the following advantages over conventional low molecular weight drug drugs.

  (1) The relationship with the target molecule is clear. The extent to which a certain drug acts on biological substances other than the target substance in the living body is very important in examining side effects and the like. However, conventional low-molecular-weight compound drugs that are thought to act mainly on proteins are used to confirm whether a protein is the target protein of a certain low-molecular-weight compound drug, and to investigate side effects that act on proteins other than the target protein. However, it is very difficult. Basically, there are not many reliable methods other than administering a low-molecular-weight compound drug directly to a living body and examining side effects and the like. This is thought to be because it is difficult to examine all the protein structures and the like in vivo. In contrast, nucleic acid drugs that are thought to act mainly on nucleic acids are much easier to identify target nucleic acids and predict side effects on nucleic acids other than target nucleic acids compared to low molecular weight compound drugs that act on proteins. It can be performed with high accuracy. Even in nucleic acid medicine, it can be considered that side effects such as homology on the gene sequence may cause functional inhibition of genes other than the target gene. However, since the gene sequence of the human genome is almost completely deciphered, Prediction is possible with high accuracy. In fact, in drug discovery using RNA interference, prediction programs (RNAi, siDirect (registered trademark), etc.) for avoiding side effects on genes that are not targets are on the market.

  (2) The rapid process from target identification in drug discovery process to acquisition of hit / lead molecules. In the manufacture of a low molecular weight compound pharmaceutical, generally, after determining a target protein, the steps of constructing an expression system for the target protein, constructing an optimal assay system, and screening from tens of thousands of compound libraries are taken. There is a need. In the process of constructing a protein expression system and the process of constructing an optimal assay system, it is necessary to find the optimum conditions according to the function of each protein type. For this reason, even these skilled engineers take several months to one year, which is very labor intensive. Further, after that, it is necessary to screen for a hit lead compound that can bind to the target protein from a compound library having at least tens of thousands of low molecular weight compounds. In contrast, in a nucleic acid drug, after determining a target gene, a hit / lead molecule of the nucleic acid drug can be designed directly based on a base sequence complementary to the base sequence of the gene. Furthermore, by performing screening from thousands of candidate nucleic acid drugs at most, molecules having sufficient effects as nucleic acid drugs can be obtained.

  Because of these advantages, the development of nucleic acid drugs is currently accelerating. For example, in February 2005, there was a report that a modified siRNA targeting age-related macular degeneration from Actuy Pharmaceuticals entered the first phase of clinical trials. This nucleic acid drug targets vascular endothelial growth factor (VEGF) to inhibit disease-causing angiogenesis in an experimental system using rats as a model.

In this way, efforts to develop nucleic acid drugs applying RNA interference technology are being actively carried out all over the world. However, there are several problems in this development process at present.
One of them is an evaluation method of RNA interference technology. In the conventional method, whether or not RNA interference has occurred is indirectly detected by examining the presence or absence of expression of a protein that is a product of the target gene. In this case, in the determination of the presence or absence of protein expression, in particular, fluorescent proteins such as GFP and proteins that catalyze a luminescent reaction such as luciferase are widely used (for example, see Non-Patent Document 4). .) However, in this method, the mRNA expression, which is the transcription product of the target gene, was degraded by RNA interference, and thus the expression of the protein was suppressed, and the case where protein synthesis was inhibited by a cause other than the degradation reaction in RNA interference. Cannot be distinguished.

  Another problem is that the efficiency of screening thousands of candidate nucleic acid drugs in a test tube is insufficient. In conventional screening methods, methods such as cell assay and computer prediction are used. However, in the cell assay, cell culture is required for screening, which is labor and costly. On the other hand, in the prediction by a computer, the prediction accuracy that an active one can be selected reliably is still low. In addition, in-vitro detection methods generally employ a large amount of candidate nucleic acid drugs in order to examine whether or not nucleic acid degradation due to RNA interference has occurred by electrophoresis of nucleic acid previously labeled with a radioisotope. Not suitable for screening performed against

  From the above two problems, in order to promote the development of nucleic acid medicine applying RNA interference technology, degradation reaction in RNA interference can be directly detected in a solution in vivo or in vitro. This suggests the importance of developing simple methods.

  On the other hand, the development of optical measurement technology in recent years is remarkable, and single molecule fluorescence analysis technology capable of measuring and analyzing fluorescence from a single fluorescent molecule is being applied in many fields. By applying single-molecule fluorescence analysis technology, for example, a molecule that becomes a substrate in a decomposition reaction is fluorescently labeled, and a fluorescent signal detected from the fluorescently labeled molecule is analyzed to detect a decomposition reaction and a change in molecular weight associated therewith. can do. To date, the methods used to detect decomposition reactions include fluorescence resonance energy transfer (FRET), fluorescence polarization (FP), fluorescence correlation spectroscopy (FCS), fluorescence cross correlation spectroscopy (FCCS), and fluorescence intensity distribution analysis. (FIDA) and the like are known.

Fluorescence resonance energy transfer (FRET) is a technique for observing the transfer of fluorescence energy between fluorescent molecules when the molecules are labeled with a plurality of fluorescent materials having different spectral characteristics and the distance between the fluorescent molecules is very close. (For example, refer nonpatent literature 5.). In general, FRET uses two types of fluorescent materials having different spectral characteristics, that is, a fluorescent material (donor) that gives fluorescent energy and a fluorescent material (acceptor) that receives the energy as excitation energy. A fluorescence resonance energy transition is observed when two fluorescent molecules mix and interact with each other and one absorption wavelength overlaps the other emission wavelength. Here, when the average interval between two fluorescent molecules is r, the energy transition efficiency is proportional to r ⁻⁶ . For this reason, when the interaction between two molecules and the mutual affinity increase, the energy transition phenomenon is enhanced, and as a result, the fluorescence emission from the acceptor increases. Therefore, conventionally, a method for detecting nucleic acid or peptide degradation by labeling a molecule as a substrate for a degradation reaction with a donor and an acceptor and detecting FRET associated with the degradation reaction is known.

  Fluorescence polarization (FP) has been used in both research and clinical applications for sensitive measurements of molecular volume and microviscosity. Molecules in solution tend to “tumble” around their various axes, and FP depends on changes in the rotational properties of molecules in solution. Specifically, larger molecules, for example larger volumes or molecular weights, tumble along fewer axes more slowly than smaller molecules. Thus, there is less movement between excitation and emission, so that the emitted light exhibits a relatively high degree of polarization. Conversely, the fluorescence emission from small fluorescent molecules that exhibit greater tumbling between excitation and emission is more multiplanar and has a lower degree of polarization. As small molecules adopt a larger or more robust conformation, their tumbling is reduced and the emitted fluorescence becomes relatively more polarized. The change in the degree of polarization of the emitted fluorescence can be measured and used as an index for increasing the size and fastness of the fluorescent molecule.

  Fluorescence correlation spectroscopy (FCS) is one method for detecting changes in molecular weight due to interaction by freely diffusing a fluorescently labeled reactant and a substance having interaction activity in a transparent medium. (For example, refer to Patent Document 3). Molecules diffusing in the medium generate fluctuations in fluorescence intensity that can be detected by the confocal optical system when passing through the focal point (very small space). Therefore, by performing autocorrelation analysis on this fluorescent signal, the molecular weight change of the reactant obtained by the interaction can be analyzed or quantified. Patent Document 3 does not disclose detection of RNA degradation reaction in RNA interference reaction.

  Fluorescence cross-correlation spectroscopy (FCCS) is a method for analyzing the interaction between molecules labeled with fluorescent substances having different spectral characteristics. In other words, the sample is irradiated with excitation light corresponding to each fluorescent substance at the same time, and the cross-correlation of the fluorescence signals detected from each molecule is analyzed to confirm whether the movements of different molecules are synchronized. can do. Thus, unlike FCS, FCCS can analyze the interaction between molecules without depending on the change in molecular size before and after the interaction. For this reason, even when the change in the molecular size before and after the interaction is small, the intermolecular interaction can be suitably analyzed.

The fluorescence intensity distribution analysis method (FIDA) is a method for analyzing the fluorescence brightness of a mixture of particles in a solution (see, for example, Patent Document 4). That is, FIDA is based on an optical and electronic configuration similar to that used for FCS analysis, but uses an algorithm different from FCS for analysis of the fluorescent signal detected from the focal point. . Specifically, this method measures a photon count histogram and determines the concentration of fluorescent particles as a function of specific brightness expressed as an average count rate per given particle. The present invention can be applied to the measurement of the concentration of various fluorescent species in different types of samples, and is a useful tool in various fields. For example, it can be applied to basic research for drug development and diagnosis. As an example, FIDA can distinguish between free fluorescently labeled antigens and fluorescently labeled antigens bound to multivalent receptors such as bivalent antibody molecules. Patent Document 4 does not disclose detection of RNA degradation reaction in RNA interference reaction.
Japanese translation of PCT publication No. 2002-516062 JP 2005-337805 A JP-T-11-502608 JP 2002-543414 A Fire, 5 others, Nature, 1998, 391, 806-811. Gil, 1 other, Apoptsis, 2000, Vol. 5, pp. 107-114. Ui-Tei, three others, FEBS letters, 2000, 479, 79-82. Elbashir, 5 people outside, Nature, 2001, 411, 494-498. Theodorus, two others, The Journal of Cell Biology, 1995, 129, 1543-1558.

  The present inventor has directly applied the RNA degradation reaction in RNA interference by applying the above-described single-molecule fluorescence analysis technology that has been conventionally used for analysis of degradation reactions and interaction reactions of nucleic acids and proteins. I thought that it could be detected efficiently.

  However, for example, when an RNA degradation reaction is to be detected using FRET, the FRET efficiency greatly depends on the molecular distance. Therefore, if the nucleic acid length of the RNA serving as the substrate is long, the FRET efficiency is poor and the cleavage occurs. It may be difficult to use for detecting the reaction. There is also a problem that the apparatus for measuring FRET is complicated.

In addition, it is known that FP is saturated with an increase in the standard polarization degree at a molecular weight of about 30 kDa. For this reason, when considering the molecular size of RNA, there is a problem that it is often impossible to distinguish between RNA before degradation and RNA after degradation.
On the other hand, in FCS, a change in molecular weight cannot be detected unless there is a difference of about 4 times in molecular weight. For this reason, there is a possibility that the RNA before degradation and the RNA after degradation cannot be distinguished depending on the RNA cleavage site due to RNA interference. As described above, in FCS, the difference in the cleavage site has a great influence on the detection ability. Therefore, FCS is not suitable for use in comparing various candidate siRNAs in parallel such as screening.

  In addition, FCCS has an advantage that analysis is possible even when the change in molecular size before and after the interaction is only about twice. However, since the fluorescence signal is detected by irradiating one observation region, which is a minute space, with excitation light having different wavelengths, the confocal region of the excitation light of each wavelength is due to axial chromatic aberration of the objective lens used. There is a serious problem that the detection sensitivity and accuracy of the target intermolecular interaction are not exactly matched and the detection sensitivity and accuracy of the target intermolecular interaction are lowered.

  An object of the present invention is to provide a method for easily and rapidly detecting an RNA cleavage reaction by siRNA used in RNA interference by applying a single molecule fluorescence analysis technique.

  As a result of intensive studies to solve the above problems, the present inventor has determined whether the single-stranded RNA has been cleaved by siRNA by using fluorescently labeled single-stranded RNA as RNA serving as a substrate for the RNA cleavage reaction. It has been found that it can be directly and efficiently detected by detecting whether or not by using a single molecule fluorescence analysis method, particularly FIDA, and completed the present invention.

That is, the present invention
(1) A method for detecting an RNA cleavage reaction by siRNA used in RNA interference, wherein whether or not the substrate RNA is cleaved by siRNA using fluorescently labeled single-stranded RNA as a substrate RNA for RNA cleavage reaction A method for detecting an RNA cleavage reaction by siRNA, characterized by detecting the above by single molecule fluorescence analysis,
(2) The method for detecting an RNA cleavage reaction by siRNA according to (1), wherein the substrate RNA is a single-stranded RNA having a 3 ′ end and a 5 ′ end labeled with a fluorescent substance,
(3) The method for detecting an RNA cleavage reaction by siRNA according to (1) or (2), wherein the single-molecule fluorescence analysis is fluorescence intensity distribution analysis (FIDA),
(4) A method for detecting an RNA cleavage reaction by siRNA used in RNA interference, wherein a single-stranded RNA having a 3 ′ end and a 5 ′ end labeled with a fluorescent substance is used as a substrate RNA for the RNA cleavage reaction A reaction step of adding and reacting siRNA and the substrate RNA to a reaction solution containing an RNA helicase and a RISC complex, and a wavelength of a wavelength capable of exciting the fluorescent substance in the reaction solution after the reaction step. A measurement process that measures the RNA cleavage reaction efficiency by irradiating light and detecting the fluorescence generated from the fluorescent substance as a fluorescence signal, and analyzing the fluorescence intensity distribution based on the fluorescence signal detected in the detection process. A method for detecting an RNA cleavage reaction with siRNA, comprising the steps of:
(5) The method for detecting an RNA cleavage reaction with siRNA according to any one of (1) to (4) above, wherein the RNA cleavage reaction with siRNA is performed in a cell or in a reaction solution,
Is to provide.

By using the method for detecting an RNA cleavage reaction by siRNA of the present invention, it is necessary to separately examine the conditions such as the length of RNA as a substrate, the size of the molecule, and the cleavage site by siRNA for each RNA. Therefore, the RNA cleavage reaction can be detected simply and rapidly.
In particular, in RNA interference, selection of a short double-stranded RNA (siRNA) of about 20 base pairs is very important for suppressing / inhibiting the function of a target gene, but the RNA cleavage reaction by the siRNA of the present invention. By using this detection method, it can be expected to save labor and time in selecting siRNA having actually high suppression efficiency from among a large number of siRNA candidate molecules.

  The method for detecting an RNA cleavage reaction by siRNA of the present invention (hereinafter sometimes referred to as the detection method of the present invention) is a method for detecting an RNA cleavage reaction by siRNA used in RNA interference, wherein Using single-stranded RNA labeled with fluorescence as the substrate RNA, whether or not the substrate RNA is cleaved by siRNA is detected by single molecule fluorescence analysis. Compared with the conventional method (for example, refer nonpatent literature 4 etc.) which measures the suppression efficiency of siRNA using the expression efficiency and functional efficiency of the protein which is an expression product of a target gene as a parameter | index, the detection method of this invention is siRNA. Since the cleavage efficiency of substrate RNA is directly measured, more reliable data can be obtained.

  The substrate RNA in the present invention is a single-stranded RNA derived from a target gene for RNA interference (a gene that is a target for suppressing or inhibiting the function of the gene or an expression product of the gene by RNA interference), and is RNA by siRNA RNA that functions as a substrate in the cleavage reaction. The single-stranded RNA derived from the target gene is not particularly limited as long as it is a single-stranded RNA transcribed from the target gene, and examples thereof include mRNA.

  The target gene may be a structural gene or a non-structural gene. In addition, the biological species from which the target gene is derived is not particularly limited, and may be any of insects such as nematodes and flies, plants, and animals. In the present invention, it is preferably a gene derived from mammals including humans.

  The substrate RNA used in the detection method of the present invention is a fluorescently labeled single-stranded RNA. Fluorescent label can distinguish between substrate RNA before cleavage and substrate RNA after cleavage by analyzing the fluorescence signal detected from fluorescent label, which can determine whether substrate RNA has been cleaved The position is not particularly limited, and can be appropriately determined in consideration of the type of single-molecule fluorescence analysis used for detection, the type of substrate RNA, the type of siRNA, and the like.

  In the detection method of the present invention, it is preferable that the 3 ′ end and 5 ′ end of the substrate RNA are labeled with a fluorescent substance. This is because the presence or absence of cleavage of the substrate RNA can be easily detected by FIDA without considering the cleavage site of the substrate RNA by siRNA because both ends of the substrate RNA are fluorescently labeled. For example, when the same kind of fluorescent substance is bound to the 3 ′ end and 5 ′ end of the substrate RNA, theoretically, the fluorescence intensity per molecule of the substrate RNA after cleavage is the substrate before cleavage. It becomes half of the fluorescence intensity per molecule of RNA. For this reason, by using FIDA that can distinguish and detect molecules having different fluorescence intensities per molecule, the cleavage reaction efficiency can be calculated by discriminating between the substrate RNA after cleavage and the substrate RNA before cleavage. .

  The fluorescent substance used for fluorescent labeling of the substrate RNA is not particularly limited, and can be appropriately determined from fluorescent substances generally used for fluorescent labeling of nucleic acids. Examples of such fluorescent substances include TAMRA, FITC (fluorescein isothiocyanate), fluorescein, rhodamine, NBD, TMR (tetramethylrhodamine), Alexa Fluor (registered trademark) (manufactured by Invitrogen), GFP (Green Fluorescent Protein). ) And YFP (Yellow Fluorescent Protein). Since the influence on the RNA cleavage reaction can be suppressed, a fluorescent substance having a relatively small molecular weight such as TAMRA, FITC, fluorescein, rhodamine, NBD, TMR, Alexa Fluor, etc. is preferable. In particular, a dye that emits fluorescence relatively stably even when continuously irradiated with light, such as TMR, is preferable. In this way, by using a fluorescent material that is difficult to fade as a label, it is possible to suppress the influence of the time and number of times of light irradiation, prevent variation for each measurement value, and obtain a more stable analysis result.

  The method for fluorescently labeling the substrate RNA is not particularly limited, and can be performed using a technique that is usually used when fluorescently labeling a nucleic acid. For example, the fluorescent material may be directly bonded to the substrate RNA, or may be indirectly bonded using the interaction between the fluorescent material and the substrate RNA. In the detection method of the present invention, a single-stranded RNA having 3 ′ and 5 ′ ends that are suitable as substrate RNA labeled with a fluorescent substance can be obtained, for example, by the following methods (A) to (C). The substrate RNA used in the present invention is not limited to those synthesized by the following (A) to (C).

・ Method (A)
First, mRNA is prepared from a target gene (DNA). For example, by using a commercially available mRNA synthesis kit such as RiboMAX System (manufactured by Promega), mRNA can be easily prepared even in vitro such as in vitro.
Next, single-stranded RNA having an arbitrary base sequence of about 10 to 20 bases in which the 5 ′ end is labeled with a fluorescent substance (5 ′ end labeled RNA), and the 3 ′ end is labeled with a fluorescent substance. Each single-stranded RNA (3 ′ end-labeled RNA) having an arbitrary base sequence having a length of about 10 to 20 bases is chemically synthesized. The base sequences of 5 ′ end-labeled RNA and 3 ′ end-labeled RNA may be the same or different. Chemical synthesis may be performed by any method known in the art, and a contract service such as Sigma Genosys may be used.

  Furthermore, mRNA prepared from the target gene, 5 ′ end-labeled RNA and 3 ′ end-labeled RNA are linked to obtain a substrate RNA in which the 3 ′ end and the 5 ′ end are labeled with a fluorescent substance, It can refine | purify by a well-known method using a difference and a difference in affinity. As an example, as schematically shown in FIG. 1, first, mRNA (1) and 5 ′ end-labeled RNA (2) are bound using an enzyme such as T4 RNA ligase and labeled with 5 ′ end. mRNA (3) is obtained. Thereafter, fractionation by molecular size is performed by gel filtration chromatography or the like, and 5 'end-labeled RNA (2) having a remarkably small molecular weight is removed. Furthermore, the carrier (4) on which the mixture of the remaining mRNA (1) and the 5 ′ end labeled mRNA (3) is fixed with a single-stranded RNA having a base sequence complementary to the 5 ′ end labeled RNA (2) Fractionation by affinity (affinity) is performed by an affinity chromatography method using the method, mRNA (1) is removed, and 5′-end labeled mRNA (3) is purified. Next, the purified 5 ′ end labeled mRNA (3) and 3 ′ end labeled RNA (5) are combined using an enzyme such as T4 RNA ligase to thereby add both end labeled mRNA (6). obtain. Thereafter, similarly, fractionation by molecular size is performed, 3 ′ end-labeled RNA (5) is removed, and then fractionation by affinity (affinity) is performed, thereby purifying both end-labeled mRNA (6 ) Is obtained.

・ Method (B)
First, in order to synthesize mRNA having an RNA aptamer that specifically binds to a fluorescent substance in both the 5 ′ side region and the 3 ′ side region of the target gene, a recombinant gene obtained by recombining the target gene (DNA) is obtained. MRNA is prepared from this recombinant gene (DNA) in the same manner as in method (A), and RNA aptamer (7) that specifically binds to a fluorescent substance is present at the 3 ′ end and 5 ′ end of mRNA (1). Aptamer-added mRNA (8) is obtained. Thereafter, as schematically shown in FIG. 2, the fluorescent substance (F) is added to the reaction solution, and after the aptamer (7) and the fluorescent substance (F) are combined, fractionation by molecular size is performed. The aptamer-added mRNA (8) bound to (F) is purified.

・ Method (C)
First, in order to synthesize mRNA having an arbitrary base sequence having a length of about 10 to 20 bases in both the 5 ′ side region and the 3 ′ side region of the target gene, a recombinant gene obtained by recombining the target gene (DNA) is obtained. . From this recombinant gene (DNA), mRNA is prepared in the same manner as in the method (A), and one having an arbitrary base sequence of about 10 to 20 bases at the 3 ′ end and 5 ′ end of mRNA (1). A sequence-added mRNA (10) having a strand RNA (9) is obtained. Thereafter, as schematically shown in FIG. 3, the reaction solution is a single-stranded RNA having a sequence complementary to the base sequence (9) and labeled at the 3 ′ end with a fluorescent substance (F). 3 ′ end-labeled RNA probe (11) and 5 ′ end-labeled RNA probe which is a single-stranded RNA having a sequence complementary to single-stranded RNA (9) and having a 5 ′ end labeled with a fluorescent substance (12) is added, and the sequence-added mRNA (10) and the 3 ′ end-labeled RNA probe (11), and the sequence-added mRNA (10) and the 5 ′ end-labeled RNA probe (12) are hybridized. The three-component complex thus formed can be purified by fractionation according to molecular size.

  In the present invention, siRNA is a double strand comprising an RNA strand (antisense strand) having a sequence complementary to part or all of the substrate RNA and an RNA strand (sense strand) having a sequence complementary thereto. RNA. The base pair length of the siRNA is not particularly limited as long as it is an effective length in RNA interference. The type and base sequence of the target gene, the biological species from which the target gene is derived, the method of introduction into cells, etc. It can be determined as appropriate in consideration. As siRNA used in RNA interference with mammals such as humans, the sequence portion complementary to the substrate RNA is preferably 15 to 25 base pairs in length.

  The siRNA used in the present invention may be designed and synthesized by any method known in the art. For example, based on the base sequence information of mRNA derived from the target gene, the base sequence of siRNA can be designed by a conventional method, and then synthesized by a known nucleic acid synthesis reaction based on the design. For example, siRNA may be synthesized independently using a commercially available synthesizer or may be synthesized using a contract service.

  The siRNA used in the present invention may have an additional base sequence other than a sequence complementary to a part or all of the substrate RNA as long as the RNA cleavage reaction against the substrate RNA is not inhibited. Alternatively, it may be a double-stranded RNA that has been modified such as 2'-O-methylation.

  The detection method of the present invention uses, for example, a reaction solution containing RNA helicase and RISC complex using single-stranded RNA labeled with a fluorescent substance at the 3 ′ end and 5 ′ end as a substrate RNA for RNA cleavage reaction. In addition, a reaction step of adding and reacting siRNA and the substrate RNA, and after the reaction step, the reaction solution is irradiated with light having a wavelength that can excite the fluorescent material, and the fluorescence generated from the fluorescent material is You may have the detection process detected as a fluorescence signal, and the measurement process which performs fluorescence intensity distribution analysis based on the fluorescence signal detected in the said detection process, and measures RNA cleavage reaction efficiency.

  First, as a reaction step, siRNA and a substrate RNA labeled with a fluorescent substance at the 3 'end and the 5' end are added to and reacted with a reaction solution containing an RNA helicase and a RISC complex. The siRNA undergoes unwinding by the RNA helicase to become single-stranded RNA, and the antisense strand and a plurality of proteins mainly form a RISC (RNA-induced silencing complex) complex. When the antisense strand of the siRNA binds to a complementary site in the substrate RNA, the substrate RNA is degraded by the ribonuclease activity of RISC.

  The reaction solution used in this reaction step is not particularly limited as long as it contains a molecule necessary for RNA cleavage reaction such as helicase or RISC, and is generally used for RNA interference. It can be prepared using the appropriate reagents and the like. A cell extract prepared from cultured cells or the like is preferable. This is because RNA interference is originally a phenomenon that occurs in a cell, and thus a cell extract contains molecules necessary for RNA interference. The cell extract can be prepared by a conventional method such as the method of Dynam et al. (See Nucleic Acids Research, 1983, Vol. 11, pp. 1475-1489).

  Moreover, this reaction process may be performed in vitro, such as in a test tube, and may be performed in a cell (in vivo). In the case of performing in vitro, for example, the substrate RNA can be cleaved by the siRNA by adding the substrate RNA to the reaction solution and then allowing the substrate RNA to react. On the other hand, in the case of performing in vivo, for example, after introducing siRNA into a cell and reacting, the substrate RNA can be further cleaved by introducing and reacting with the substrate RNA. In addition, siRNA or substrate RNA may be introduced into cells by any technique known in the art. For example, by using a commercially available nucleic acid introduction reagent such as lipofectamine 2000 (manufactured by Invtrogen), siRNA or substrate RNA can be easily introduced into cells.

  The reaction time can be appropriately determined in consideration of the type of reaction solution, the activity of helicase, RISC, etc. in the reaction solution, the concentration of substrate RNA or siRNA, and the like. The reaction temperature can be appropriately set within a temperature range suitable for the activity of helicase, RISC, etc. in the reaction solution. For example, when the reaction solution is a solution obtained by adding substrate RNA to a cell culture solution prepared from human-derived cultured cells, after adding siRNA to the reaction solution and reacting at 37 ° C. for 15 minutes or more Furthermore, substrate RNA can be added and reacted at 37 ° C. for about 2 to 6 hours. On the other hand, when the reaction step is performed in human-derived cultured cells, for example, siRNA is introduced into a cell culture cultured at 37 ° C. using a nucleic acid introduction reagent, and after 48 to 96 hours have elapsed after introduction, Substrate RNA can be introduced using the introduction reagent and further cultured for 48 to 96 hours.

  Next, after the reaction step, as a detection step, the reaction solution is irradiated with light having a wavelength that can excite the fluorescent substance used for labeling the substrate RNA, and fluorescence generated from the fluorescent substance is detected as a fluorescent signal. Specifically, the reaction solution is placed in a fluorometer having a confocal optical system, and a fluorescent signal can be detected by a conventional method. In addition, when the reaction step is performed in a cell, the cell can be placed in a fluorescence microscope having a confocal optical system, and a fluorescence signal can be detected by a conventional method. Single molecule fluorescence analysis methods such as FIDA generally use a confocal optical system. However, fluorescence intensity distribution analysis can be performed by obtaining a fluorescence signal derived from a single molecule. The optical system is not particularly limited, and other optical systems other than the confocal optical system can be used.

  The measurement condition of the fluorescence signal can be appropriately determined according to the specifications of the fluorometer and the fluorescence microscope to be used. For example, it is performed about 5 times in 15 seconds per sample. The measurement time may be longer, and the number of times may be longer. However, depending on the type of apparatus used for detection, if the measurement time is as short as 10 seconds or less, or if the number of measurements is only about once, the reproducibility and reliability of data may be reduced. There is. Further, by scanning the focal position with respect to the sample for measuring the fluorescence signal, information (fluorescence signal) can be obtained from a larger number of molecules, and statistical accuracy can be improved.

  Thereafter, as a measurement step, fluorescence intensity distribution analysis is performed based on the detected fluorescence signal, and the RNA cleavage reaction efficiency is measured. That is, the fluorescence intensity distribution analysis is performed on the detected fluorescence signal data, whereby the fluorescence intensity of the fluorescent molecule and the concentration in the detection region can be calculated. Specifically, from the fluorescence intensity per molecule, it is determined whether the fluorescent molecule is a cleaved substrate RNA or an uncleaved substrate RNA, and the ratio of the number of these molecules is calculated.

  The efficiency of the RNA cleavage reaction can be judged from a decrease in fluorescence intensity or an increase in the concentration of molecules with low fluorescence intensity. FIG. 4B is a graph showing changes in the number of photons (Number of photons) and the probability (Probability) before and after the RNA cleavage reaction. As shown in FIG. 4 (A), theoretically, the fluorescence intensity of the cleaved substrate RNA is approximately half the fluorescence intensity of the uncleaved substrate RNA. Furthermore, the number of fluorescent molecules is doubled by RNA cleavage. That is, the higher the ratio of the number of molecules with low fluorescence intensity, the higher the reaction efficiency of the RNA cleavage reaction with siRNA.

  Hereinafter, one embodiment of the present invention in each of the case where the reaction step is performed inside the cell and the case where the reaction step is performed outside the cell will be described. In addition, this invention is not limited to the following embodiments.

[When reaction process is performed in cells]
1. A target gene (DNA) for which the efficiency of RNA interference is to be detected is prepared. By using RiboMAX System (manufactured by Promega) or the like, mRNA is expressed from the target gene (DNA) in a test tube.
2. Both ends of mRNA are fluorescently labeled by any one of the above methods (A), (B), and (C).
3. Using a nucleic acid introduction reagent such as lipofectamine 2000 (manufactured by Invtrogen), a siRNA having a length of about 20 base pairs targeting the target gene is introduced into the cultured cells to cause RNA interference. After introduction of siRNA, the fluorescence-labeled mRNA synthesized in 2 above is introduced into the cultured cells after culturing for 48 to 96 hours using a nucleic acid introduction reagent, and further cultured for 48 to 96 hours.
4). A fluorescence pattern is directly detected from the cultured cells cultured for 48 to 96 hours after introduction using a fluorometer using a confocal optical system and recorded in a recording unit or the like. When detecting the fluorescence pattern, the focal position of the cultured cell (sample) is scanned to obtain information (fluorescence signal) from a larger number of molecules to improve statistical accuracy and at the same time spatial resolution efficiency It is also possible to acquire the information.
5). FIDA is performed on the recorded fluorescence pattern data, the translation time of the fluorescent molecule is obtained, and the fluorescence intensity and concentration of each measured fluorescent molecule are calculated. The efficiency of the RNA cleavage reaction can be judged from a decrease in fluorescence intensity or an increase in the concentration of fluorescent molecules.

[When reaction process is performed outside the cell]
By using siRNA having a sequence that directly becomes a nucleic acid drug candidate, this is a suitable method for screening candidate nucleic acid drugs.
1. A target gene (DNA) for which the efficiency of RNA interference is to be detected is prepared. By using RiboMAX System (manufactured by Promega) or the like, mRNA is expressed from the target gene (DNA) in a test tube.
2. Both ends of mRNA are fluorescently labeled by any one of the above methods (A), (B), and (C).
3. A cell extract is prepared from cultured cells by a conventional method.
4). To the cell extract prepared in 3 above, siRNA having a length of about 20 base pairs targeting the target gene is added and mixed, and incubated at 37 ° C. for about 15 minutes or longer.
5). The following reagents are added and mixed to a predetermined concentration: 10 mM HEPES-KOH (pH 7.9), 0.5 mM DTT, 1.5 mM MgCl ₂ , 100 mM KCl, 1 mM ATP, 0.2 mM GTP, 10 U / Ml Rnasin (manufactured by Promega), the fluorescently labeled mRNA synthesized in the above 2 prepared to a concentration of 1 nM to 10 nM, the cell extract of the above 4 prepared to a volume of 50% of the total.
6). The reaction solution prepared in 5 above is incubated at 37 ° C. for about 2 to 6 hours.
7). After the reaction, the reaction solution is placed in a fluorimeter using a confocal optical system, and a fluorescence pattern is detected and recorded in a recording unit or the like. When detecting the fluorescence pattern, the focal position of the sample (reaction solution) is scanned to obtain information (fluorescence signal) from a larger number of molecules to improve statistical accuracy and at the same time spatial resolution efficiency It is also possible to acquire the information.
8). FIDA is performed on the recorded fluorescence pattern data, the translation time of the fluorescent molecule is obtained, and the fluorescence intensity and concentration of each measured fluorescent molecule are calculated. The efficiency of the RNA cleavage reaction can be judged from a decrease in fluorescence intensity or an increase in the concentration of fluorescent molecules.

  By using the method for detecting an RNA cleavage reaction by siRNA of the present invention, an RNA cleavage reaction can be detected easily and rapidly. Therefore, siRNA having a high RNA interference effect can be easily screened from a large number of candidate molecules. It can be used not only in the field of academic research, but also in the field of development of nucleic acid drugs applying siRNA.

It is the figure which showed typically the method (A) which manufactures fluorescent-labeled substrate RNA. It is the figure which showed typically the method (B) which manufactures fluorescence-labeled substrate RNA. It is the figure which showed typically the method (C) which manufactures the fluorescently labeled substrate RNA. FIG. 4A schematically shows a reaction in which the substrate RNA (both end-labeled mRNA (6)) labeled with a fluorescent substance at the 3 ′ end and the 5 ′ end is cleaved by the RNA cleaving reaction. It is. FIG. 4B shows changes in the number of photons (Number of photons) and the probability (Probability) before and after the RNA cleavage reaction.

Explanation of symbols

  1 ... mRNA, 2 ... 5 'end labeled RNA, 3 ... 5' end labeled mRNA, 4 ... carrier, 5 ... 3 'end labeled RNA, 6 ... both ends labeled mRNA, 7 ... specific binding to fluorescent substance RNA aptamer, 8 ... aptamer added mRNA, 9 ... single stranded RNA, 10 ... sequence added mRNA, 11 ... 3 'end labeled RNA probe, 12 ... 5' end labeled RNA probe.

Claims (5)

A method for detecting an RNA cleavage reaction by siRNA used in RNA interference,
RNA cleavage by siRNA, wherein single-stranded RNA fluorescently labeled as a substrate RNA for RNA cleavage reaction is used to detect whether or not the substrate RNA has been cleaved by siRNA by single molecule fluorescence analysis Reaction detection method.   2. The method for detecting an RNA cleavage reaction by siRNA according to claim 1, wherein the substrate RNA is a single-stranded RNA having a 3 'end and a 5' end labeled with a fluorescent substance.   The method for detecting an RNA cleavage reaction by siRNA according to claim 1 or 2, wherein the single molecule fluorescence analysis method is a fluorescence intensity distribution analysis method (FIDA). A method for detecting an RNA cleavage reaction by siRNA used in RNA interference,
As the substrate RNA for the RNA cleavage reaction, single-stranded RNA having 3 ′ end and 5 ′ end labeled with a fluorescent substance is used,
A reaction step of adding and reacting siRNA and the substrate RNA to a reaction solution containing RNA helicase and RISC complex;
After the reaction step, the reaction solution is irradiated with light having a wavelength that can excite the fluorescent substance, and the fluorescence generated from the fluorescent substance is detected as a fluorescent signal;
A measurement step of performing fluorescence intensity distribution analysis based on the fluorescence signal detected in the detection step, and measuring RNA cleavage reaction efficiency;
A method for detecting an RNA cleavage reaction by siRNA, comprising:   The method for detecting an RNA cleavage reaction by siRNA according to any one of claims 1 to 4, wherein the RNA cleavage reaction by siRNA is carried out in a cell or in a reaction solution.

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