JP2010151550A - METHOD FOR EVALUATING GENE EXPRESSION INHIBITING EFFECT OF siRNA - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for quickly and conveniently evaluating a gene expression inhibiting effect by a siRNA. <P>SOLUTION: The method for evaluating the gene expression inhibiting effect of the siRNA includes: the process (a) for preparing a fluorescently-labeled siRNA in which one RNA strand as a sense strand or an antisense strand in the siRNA is labeled by a fluorescent material; the process (b) for irradiating a reaction solution containing the fluorescently-labeled siRNA with excitation light, detecting a fluorescence as a fluorescent signal generated from the fluorescent material, and analyzing it by a unimolecular fluorescence analysis method; the process (c) for irradiating the reaction solution with excitation light, detecting the fluorescence as the fluorescent signal generated from the fluorescent material, and analyzing it by the unimolecular fluorescence analysis method after the fluorescently-labeled siRNA is added to the reaction solution containing a RNA helicase or a RISC and an ATP, and reacted; and the process (d) for comparing analysis results obtained from the processes (b), (c), and evaluating the gene expression inhibiting effect of the siRNA. <P>COPYRIGHT: (C)2010,JPO&INPIT

Description

  The present invention relates to a method for evaluating the gene expression inhibitory effect of siRNA used in RNA interference by analysis at a single molecule level.

  In RNA interference, double-stranded RNA is incorporated into cells of various living organisms such as nematodes, flies, plants, animals, etc., and mRNA having a base sequence complementary to the double-stranded RNA is degraded. As a result, this is a phenomenon in which arbitrary gene expression is suppressed. The cleaved long (generally 200 base pairs or more) double-stranded RNA is converted into a short double-stranded RNA (siRNA: short interfering RNA) of 21 to 25 base pairs by Dicer, a kind of ribonuclease, in the cell. Disconnected. This siRNA, which is a short double-stranded RNA, is unwound by helicase in the cytoplasm and becomes single-stranded, and this single-stranded RNA and RISC (RNA induced silencing complex) composed of a plurality of proteins are RISC complex. Form the body. Thereafter, single-stranded RNA in the RISC complex and mRNA having a complementary base sequence are further incorporated into the RISC complex, and the mRNA is degraded by the ribonuclease activity of RISC, and gene silencing (gene silencing) is performed. Expression suppression) occurs. RNA interference eliminates the need for equipment and time required for the production of knockout mice by homologous recombination, and allows easy knockdown of target gene mRNAs in cultured cell and model animal experimental systems. Interference is used for gene function analysis and target gene screening.

  However, in mammals such as humans, long double-stranded RNAs cause an interferon response, and thus long double-stranded RNAs cannot be directly introduced into cells. For this reason, for example, there is a technique of introducing it into cells as an expression vector incorporating double-stranded RNA. In this technique, an expression vector in which a nucleotide sequence of shRNA (short hairpin RNA) is inserted downstream of a promoter sequence is used, and siRNA is expressed through shRNA processing in a cell to cause RNA interference. In addition, since it takes a certain amount of time to construct an expression vector, when it is desired to confirm the effect of suppressing gene expression more quickly and simply, a method of directly introducing a chemically synthesized siRNA of about 21 base pairs into a cell. Is used.

  In RNA interference, a synthetic siRNA introduced into a cell interacts with a target mRNA and suppresses translation of the target protein by cleaving this. More specifically, siRNA introduced into a cell interacts with a RISC loading complex such as Dicer and is carried to RISC. RISC cleaves double-stranded siRNA and takes up the resulting single-stranded RNA. At this time, the RISC-antisense strand complex is formed by incorporating the antisense strand into RISC. The complex recognizes and binds to the target mRNA having a base sequence complementary to the antisense strand, and the mRNA is cleaved by RISC to suppress the expression of the target gene (target gene). (For example, refer nonpatent literature 1.)

  In addition, due to recent advances in single-molecule fluorescence analysis technology, it has become possible to directly observe the behavior of siRNA introduced into cells. For example, after introducing fluorescently labeled siRNA into a cell, the behavior of the siRNA is observed using fluorescence correlation spectroscopy (FCS) or fluorescence cross-correlation spectroscopy (FCCS). can do. (For example, refer nonpatent literature 2.) In addition, the state and behavior of siRNA introduce | transduced in the cell can also be observed using fluorescence resonance energy transfer (Fluorescence resonance energy transfer: FRET). (For example, refer nonpatent literature 3.)

  As a technique for confirming the effect of suppressing the expression of a target gene by introducing siRNA into a cell, a technique using real-time PCR is generally used. In this method, first, cells into which siRNA has been introduced are cultured for a certain period. MRNA is extracted from the cultured cells, and cDNA is prepared by reverse transcription. Real-time PCR is performed using the obtained cDNA, and the amount of mRNA derived from the target gene is quantitatively measured to evaluate whether the expression of the target gene is suppressed. When the expression of the target gene is suppressed, the amount of mRNA derived from the target gene is decreased. For this reason, it can be evaluated that siRNA having a higher tendency to decrease the amount of mRNA has a higher effect of suppressing the expression of the target gene as compared with that before introduction of siRNA into the cell.

  On the other hand, in the process up to suppression of target gene expression by siRNA, when the siRNA introduced into the cell is seeded back by helicase and the single-stranded RNA generated by cleavage is incorporated into RISC, not the antisense strand but the sense strand May be taken in and a RISC-sense strand complex may be formed. In this case, the mRNA having a base sequence complementary to the sense strand is cleaved by the RISC-sense strand complex, and the expression of a gene different from the target gene is suppressed. Such an effect of suppressing the expression of genes other than the target gene is referred to as an off-target effect. In addition, when a RISC-antisense strand complex is formed, due to a mismatch, mRNA having a sequence that is non-complementary to the antisense strand is recognized and cleaved, and expression of a gene other than the target gene may occur. It may be suppressed. Such an effect is also known as an off-target effect.

In order to avoid such an off-target effect, some contrivances have been made regarding the design of siRNA to be introduced. In order to preferentially form a RISC-antisense strand complex, it is preferable to perform cleavage of siRNA by RNA helicase constituting RISC from the sense strand side of siRNA. Therefore, for example, in the design of siRNA, the nucleotide sequences at the 3 ′ end of the antisense strand and the 5 ′ end of the sense strand are changed to a sequence rich in guanine and cytosine (G / C rich sequence), while the antisense strand By making the base sequence of the 5 ′ end and the 3 ′ end of the sense strand into a sequence rich in adenine and uracil (A / U rich sequence), the binding strength between the antisense strand and the sense strand is reduced to 5 ′ of the sense strand. There is a method in which the end side is weaker than the 3 ′ end side of the sense strand, and cleavage by RISC is preferentially performed from the sense strand side. In addition, there is a method in which cleavage by RISC is preferentially performed from the sense strand side by modifying the base of the sense strand of siRNA.
Martinez, four others, Cell, 2002, 110, 563-574. Ohrt, 7 others, Nucleic Acids Research, 2008, Vol. 36, No. 20, pages 6439-6449. Jarve, 11 others, Nucleic Acids Research, 2007, Vol. 35, No. 18, e124.

  In any case, whether or not a certain siRNA has an off-target effect is determined by introducing siRNA into the cell, culturing the cell, extracting mRNA, cDNA synthesis, quantifying mRNA by real-time PCR, or expression product ( A series of steps such as analysis of protein) is required. These operations are very complicated, and it takes 2-3 days for the steps from siRNA introduction to cell culture, and the subsequent operations require about half a day to 1 day.

On the other hand, the target gene expression suppression effect and off-target effect of siRNA mainly depend on the design (base sequence and modification) of siRNA. For this reason, for example, when siRNA synthesized for RNA interference with a certain target gene has a large influence of off-target effect, a new siRNA is designed, and this new siRNA Then, the above series of confirmation steps is performed again to evaluate the effect. This is repeated until a good siRNA having a high target gene expression suppressing effect and a low off target effect is obtained. In general, the above steps are repeated for a plurality of candidate siRNAs, the effects of each are evaluated, and an optimal siRNA is selected.
However, there is a problem that repeating the above steps for a plurality of siRNAs requires much time and labor. Therefore, development of a method capable of more quickly and simply evaluating the target gene expression suppression effect and off-target effect of siRNA is required.

  An object of this invention is to provide the method of evaluating the gene expression suppression effect by siRNA used in RNA interference rapidly and simply.

  As a result of diligent research to solve the above problems, the present inventor evaluated the effect of siRNA on the suppression of gene expression of a target gene by examining the cleavage of siRNA and the state of incorporation of the resulting antisense strand into RISC. And by applying single-molecule fluorescence analysis technology, the effect can be evaluated without requiring a series of complicated operations such as mRNA extraction, cDNA synthesis, and mRNA quantification by real-time PCR. The headline and the present invention were completed.

That is, the present invention
(1) A method for evaluating the effect of suppressing gene expression by siRNA, wherein (a) a fluorescently labeled siRNA in which either the sense strand or the antisense strand of siRNA to be evaluated is labeled with a fluorescent substance And (b) irradiating the reaction solution containing the fluorescent-labeled siRNA with light having a wavelength that can excite the fluorescent substance, and detecting the fluorescence generated from the fluorescent substance as a fluorescent signal. A step of analyzing a fluorescence signal by a single molecule fluorescence analysis method, and (c) a reaction solution containing RNA helicase or RISC and ATP, the fluorescence labeled siRNA is added and reacted, and then the reaction solution is mixed with the reaction solution. Irradiate light with a wavelength that can excite the fluorescent substance, detect the fluorescence generated from the fluorescent substance as a fluorescent signal, and match the detected fluorescent signal. A step of analyzing by molecular fluorescence analysis, and (d) comparing the analysis result obtained in the step (b) with the analysis result obtained in the step (c), and evaluating the gene expression suppression effect of the siRNA SiRNA gene expression inhibitory effect evaluation method, characterized by comprising:
(2) There are two or more types of siRNA to be evaluated, and the steps (a) to (d) are performed on each siRNA. The method for evaluating the siRNA gene expression inhibitory effect according to (1),
(3) A method for evaluating the effect of suppressing gene expression by siRNA, wherein (a ′) a fluorescent label in which either the sense strand or the antisense strand of siRNA to be evaluated is labeled with a fluorescent substance a step of preparing siRNA; (b ′) after introducing into the cell siRNA that does not undergo unwinding of the RNA helicase labeled with the fluorescent substance, or siRNA that is not taken up by RISC labeled with the fluorescent substance; Irradiating the cell with light having a wavelength capable of exciting the fluorescent substance, detecting fluorescence generated from the fluorescent substance as a fluorescent signal, and analyzing the detected fluorescent signal by a single molecule fluorescence analysis method; , (C ′) after introducing and reacting the fluorescently labeled siRNA into a cell, the cell has a wavelength that can excite the fluorescent substance. A step of irradiating light, detecting fluorescence generated from the fluorescent substance as a fluorescence signal, and analyzing the detected fluorescence signal by a single molecule fluorescence analysis method; and (d ′) obtained in the step (b ′). Comparing the analysis result with the analysis result obtained in the step (c ′) and evaluating the gene expression inhibitory effect of the siRNA, and a method for evaluating the siRNA gene expression inhibitory effect,
(4) The method for evaluating the siRNA gene expression inhibitory effect according to any one of (1) to (3), wherein the fluorescently labeled siRNA has a sense strand labeled with a fluorescent substance,
(5) The single-molecule fluorescence analysis method includes fluorescence correlation spectroscopy (FCS), fluorescence intensity distribution analysis method (Fluorescence-Intensity Distribution Analysis: FIDA), and fluorescence polarization analysis method (FIDA) The method for evaluating the effect of suppressing gene expression of siRNA according to any one of (1) to (4), wherein the method is selected from the group consisting of PO),
Is to provide.

  The method for evaluating the gene expression suppression effect of siRNA of the present invention is to confirm the degree of off-target effect by measuring the state of siRNA incorporation into RISC by single-molecule fluorescence analysis using fluorescently labeled siRNA. Can do. Therefore, by using the siRNA gene expression suppression effect evaluation method of the present invention, the siRNA gene expression suppression effect requires a series of complicated operations such as mRNA extraction, cDNA synthesis, and mRNA quantification by real-time PCR. And can be evaluated quickly and easily.

The method for evaluating the gene expression suppression effect of siRNA of the present invention is a method for evaluating the gene expression suppression effect of siRNA, which comprises the following steps.
(A) preparing a fluorescently labeled siRNA in which either one of the sense strand or the antisense strand of the siRNA to be evaluated is labeled with a fluorescent substance;
(B) The reaction solution containing the fluorescently labeled siRNA 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, and the detected fluorescent signal is single-molecule fluorescent. A process of analyzing by an analysis method;
(C) After reacting the reaction solution containing RNA helicase or RISC and ATP by adding the fluorescent-labeled siRNA, the reaction solution is irradiated with light having a wavelength capable of exciting the fluorescent substance, and Detecting fluorescence generated from a fluorescent substance as a fluorescence signal, and analyzing the detected fluorescence signal by a single molecule fluorescence analysis method;
(D) A step of comparing the analysis result obtained in the step (b) with the analysis result obtained in the step (c) to evaluate the gene expression inhibitory effect of the siRNA.

  By analyzing and comparing the state of fluorescently labeled siRNA before and after being affected by RISC (before RNA interference) and after being affected by RISC and the like, fluorescence-labeled siRNA It is possible to confirm which of the sense strand and the antisense strand constituting the RNA has been incorporated into RISC. When the antisense strand is preferentially incorporated into RISC, the siRNA should be evaluated as having a small off-target effect, a large gene expression inhibitory effect (on-target effect) of the target gene, and a high gene expression inhibitory effect. Can do. On the other hand, when the sense strand is preferentially incorporated into RISC, the siRNA can be evaluated as having a large off-target effect, a small on-target effect, and a low gene expression suppressing effect.

Hereinafter, it demonstrates for every process.
First, as step (a), a fluorescently labeled siRNA in which either the sense strand or the antisense strand of the siRNA to be evaluated is labeled with a fluorescent substance is prepared.

  The siRNA used in the present invention is a part of mRNA (target gene-derived mRNA) transcribed from a target gene (a gene that is a target of suppressing or inhibiting the function of the gene or an expression product of the gene by RNA interference) This is a double-stranded RNA comprising a sense strand that is a single-stranded RNA having a base sequence that is homologous to the whole and an antisense strand that is a single-stranded RNA having a base sequence complementary to the sense strand. The sense strand may have an additional base sequence in addition to the base sequence homologous to the target gene-derived mRNA, and may be modified by phosphorylation or 2′-O-methylation. It may be. Similarly, the antisense strand may be modified.

  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 base pair length of siRNA used in the present invention is not particularly limited as long as it is effective in RNA interference. The type and base sequence of the target gene, the biological species from which the target gene is derived, cells, etc. It can be determined appropriately in consideration of the introduction method and the like. As siRNA used in RNA interference with mammals such as humans, the sequence portion complementary to the target gene-derived mRNA 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. For example, siRNA can also be synthesized by separately synthesizing the sense strand and the antisense strand and then annealing them. It should be noted that after annealing, the remaining single-stranded RNA is preferably removed by exonuclease treatment or the like.

  The siRNA used in the present invention is a fluorescently labeled siRNA in which either the sense strand or the antisense strand is labeled with a fluorescent substance. The site in the RNA strand labeled with a fluorescent substance is a region that does not inhibit a series of RNA interference processes such as unwinding by helicase, cleavage by RISC, recognition of target gene-derived mRNA, and cleavage of target gene-derived mRNA. If it is, it will not specifically limit and it can determine suitably considering the method of a fluorescent label, the kind of the single molecule fluorescence analysis method etc. which are used. Specifically, for example, by synthesizing a single-stranded RNA using a primer in which a fluorescent substance is bound to the 5 ′ end, a sense strand or an antisense strand in which the 5 ′ end is fluorescently labeled can be obtained. it can.

  The fluorescent material used for siRNA fluorescent labeling is not particularly limited, and can be appropriately determined from fluorescent materials generally used for nucleic acid fluorescent labeling. Examples of such fluorescent substances include TAMRA, rhodamine green, Alexa Fluor (registered trademark) (manufactured by Invitrogen), ATTO dye series (manufactured by ATTO-TEC) such as ATTO 663, FITC (fluorescein isothiocyanate), fluorescein. , Rhodamine, NBD, TMR (tetramethylrhodamine) and the like. In particular, a dye that emits fluorescence relatively stably even when continuously irradiated with light, such as TAMRA and 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.

  In the present invention, it is preferable that the sense strand is a fluorescence-labeled siRNA in which the sense strand is labeled with a fluorescent substance. In RNA interference, in order to obtain an effect of suppressing the expression of a target gene, it is necessary to preferentially incorporate an antisense strand into RISC. The sense strand that has become single-stranded RNA is cleaved and subdivided by exonuclease or the like. Thus, when the on-target effect is achieved, the sense strand has a greater change in the size of the molecule in the RNA interference process than the antisense strand. For this reason, when the sense strand is fluorescently labeled, it is relatively easy to discriminate whether the RNA strand incorporated into RISC is the sense strand or the antisense strand by single molecule fluorescence analysis rather than when the antisense strand is fluorescently labeled. This is because it is easy.

  Next, as a step (b), the reaction solution containing the fluorescently labeled siRNA was irradiated with light having a wavelength capable of exciting the fluorescent substance, and the fluorescence generated from the fluorescent substance was detected as a fluorescent signal, and detected. The fluorescence signal is analyzed by single molecule fluorescence analysis. By this step, the state of the fluorescently labeled siRNA before being affected by RISC or the like (before RNA interference) can be examined.

  The reaction solution used in the step (b) is not particularly limited as long as it is a solution containing fluorescently labeled siRNA and does not cause RNA interference, and is generally used in preparation of nucleic acid samples and the like. It can be used by appropriately selecting from solutions. The “reaction solution that does not cause RNA interference” refers to a component essential for RNA interference, particularly cleavage of siRNA and incorporation into RISC, such as a reaction solution that does not contain RNA helicase or a reaction solution that does not contain RISC. The reaction solution which does not contain at least 1 sort (s) of is meant. In addition, it is also preferable that the reaction solution does not contain ATP. This is because, in general, in order to rewind siRNA, RISC needs to be activated by ATP.

  In the present invention, the reaction solution used in step (b) is preferably a solution obtained by adding fluorescently labeled siRNA to a buffer. This is because depending on the type of fluorescent substance used for siRNA labeling, it may be easily affected by pH and the like. Examples of the buffer include a phosphate buffer having a pH of 7 to 8, a 10-200 mM Tris buffer, a HEPES buffer, a Hunks buffer, and the like. In addition, it is also preferable that an RNA-degrading enzyme inhibitor such as RNasin is added to the reaction solution used in step (b).

  Furthermore, as a step (c), after adding the fluorescence labeled siRNA to a reaction solution containing RNA helicase or RISC and ATP and reacting, light having a wavelength capable of exciting the fluorescent substance in the reaction solution. , The fluorescence generated from the fluorescent substance is detected as a fluorescence signal, and the detected fluorescence signal is analyzed by a single molecule fluorescence analysis method. By this step, the state of the fluorescently labeled siRNA after being affected by RISC or the like can be examined.

  The reaction solution used in step (c) should be the same as the reaction solution listed as the reaction solution used in step (b) except that it contains both RNA helicase or RISC and ATP. Can do. In the present invention, the reaction solution used in step (c) preferably has the same composition as the reaction solution used in step (b) except that it contains both RNA helicase or RISC and ATP.

  RISC is a complex composed of a plurality of proteins such as Argonaut 2 and RNA helicase. As the RISC used in the present invention, those extracted and purified from cultured cells by a conventional method can be used.

  In the present invention, the siRNA cleavage and the incorporation of the resulting single-stranded RNA into RISC are observed to confirm the degree of siRNA off-target effect and on-target effect, thereby suppressing gene expression. To evaluate. For this reason, in particular, only an RNA helicase having a function of directly interacting with siRNA to unwind and cleave the double strand or a RISC unit containing the RNA helicase may be added to the reaction solution. As the RNA helicase or RISC unit containing the RNA helicase, those extracted and purified from cultured cells by a conventional method can be used as in RISC. As the RNA helicase, a commercially available enzyme may be used.

  In addition, what added the cell extract to the reaction solution used in the step (b) may be a reaction solution containing RNA helicase or RISC, ATP, and fluorescently labeled siRNA. This is because the cell extract usually contains RISC, RNA helicase, ATP, and the like. The cell extract can be prepared by a conventional method such as the method of Dignam et al. (Nucleic Acids Research, 1983, Vol. 11, pages 1475-1489).

  In addition, you may add RNA helicase or RISC to the reaction solution which detected the fluorescence signal in the process (b), and you may perform a process (c), and the reaction solution in a process (b) and the process (c) It may be prepared separately from the reaction solution. In the latter case, step (c) may be performed after step (b), step (b) may be performed after step (c), or both steps may be performed simultaneously.

  Further, the reaction solution in steps (b) and (c) may be an extracellular solution or an intracellular solution. That is, the reaction in steps (b) and (c) and the detection of a fluorescent signal may be performed in vivo. By observing the state of siRNA in the cell, it is possible to evaluate the effect of suppressing siRNA gene expression in a more physiological environment.

  For example, a cell that does not express RNA helicase or RISC is used as the reaction solution in step (b), and a cell that expresses RNA helicase or RISC is used as the reaction solution in step (c). As in the case of using the extracellular solution, the state of the fluorescence-labeled siRNA before being affected by RISC or the like (before RNA interference) and after being affected by RISC or the like can be observed. . Examples of cells that do not express RNA helicase or RISC include Dhamacon, RISC knockdown cells, and cells that have been knocked down by narrowing down to RNA helicase that constitutes RISC. On the other hand, as cells expressing RNA helicase or RISC, commonly used cultured cells can be used. In general, the nucleic acid introduction efficiency tends to vary depending on the type of cultured cell line. For this reason, it is preferable that the cell used in the step (b) is a knockdown cell or the like prepared from the same cell line as the cell used in the step (c).

  As the reaction solution in the step (b), an extracellular solution which is similar to the cytoplasmic environment is used, and as the reaction solution in the step (c), a cell expressing RNA helicase or RISC is used. Also good. Examples of such an extracellular solution include a BSA (bovine serum albumin) solution having a high concentration (300 to 400 mg / ml). Thus, even if it is difficult to obtain cells that do not express RNA helicase or RISC by making the reaction solution in step (b) an extracellular solution, The state of siRNA in an environment where RNA interference occurs can be observed.

  The introduction of the fluorescently labeled siRNA into the cells in the steps (b) and (c) may be performed by any technique known in the art. Examples of the method include a method using a lipid such as lipofectin, an electric method such as electroporation, a microinjection method, and the like. In addition, it can be carried out simply by using a commercially available nucleic acid introduction reagent such as lipofectamine 2000 (manufactured by Invtrogen). For example, a fluorescent-labeled siRNA prepared with a culture solution and lipid prepared so as to have a final concentration of 10 nM is introduced into cultured cells. After the introduction process, the state of introduction can be confirmed by observing fluorescence at a single molecule level by using a total reflection fluorescence microscope or the like.

  In the step (c), the fluorescent labeled siRNA is unwound by the RNA helicase and cleaved during the reaction time in which the fluorescent labeled siRNA is added to the reaction solution containing RNA helicase or RISC and ATP. It is not particularly limited as long as the time is sufficient, and can be appropriately determined in consideration of the type of reaction solution, reaction temperature, activity of RNA helicase or RISC to be used, concentration of fluorescently labeled siRNA, and the like. . For example, when the reaction solution is an extracellular solution (when performed in vitro), the reaction time is preferably about 10 minutes to 2 hours, more preferably about 15 minutes to 1 hour at 37 ° C. On the other hand, the reaction time when the reaction solution is an intracellular solution (when performed in vivo) is preferably about 1 to 10 hours and preferably about 1 to 5 hours after introduction of the fluorescently labeled siRNA under 37 ° C. culture conditions. Is preferred.

  However, when performed in vivo, it is preferable to detect the fluorescence signal over time after the introduction of the fluorescence-labeled siRNA. In the cell, the introduced siRNA is judged to be a foreign substance, and is metabolized by being decomposed by a double-stranded RNA-specific RNase or the like existing in the cell or discharged to the outside by exocytosis. There is a case. For this reason, not all siRNAs introduced into cells are processed by interaction with Dicer and RISC and contribute to the degradation of mRNA of the target gene, but the fluorescence signal is detected immediately after introduction. Thus, it is possible to know the status of introduction of fluorescently labeled siRNA into cells, the status of degradation in cells, and the like. When detecting a fluorescent signal over time, the timing for starting detection is not particularly limited, and may be immediately after the introduction of the fluorescently labeled siRNA. You may decide. For example, when the introduction of fluorescently labeled siRNA is performed by electroporation, the addition of lipofectin to the cell culture medium is performed when lipofection using lipofectin is performed from about 30 minutes after the pulse treatment. The fluorescent signal may be detected over time from the point of about 1 hour after the treatment.

  For example, in steps (b) and (c), after introducing a fluorescently labeled siRNA into a cell, the timing at which the fluorescently labeled siRNA is taken into RISC can be known by detecting the fluorescent signal over time. . In addition, it is possible to clarify the behavior when the fluorescence-labeled siRNA is not incorporated into RISC, such as whether or not degradation by exonuclease is performed.

  In addition, when step (c) is performed in vivo, fluorescently labeled siRNA that has been confirmed to be incorporated into RISC by antisense strands that have been unwound by RNA helicase or cleaved to form single-stranded RNA. May be a positive control siRNA. Specifically, after introducing and reacting the positive control siRNA to the cells into which the fluorescently labeled siRNA is introduced in the step (c), light having an appropriate wavelength is irradiated in the same manner as in the step (c). Then, the detected fluorescence signal is analyzed by single molecule fluorescence analysis. The fluorescent material for labeling the positive control siRNA may be the same type of fluorescent material as the fluorescently labeled siRNA introduced in step (c) or a different type of fluorescent material. As such a positive control siRNA, for example, a fluorescently labeled siRNA generally used as a positive control for RNA interference, such as positive control GAPDH siRNA (Ambion), can be used.

  Specifically, in the steps (b) and (c), the fluorescence signal is detected by installing the reaction solution in a fluorometer having a confocal optical system and detecting the fluorescence signal by a conventional method. be able to. When the reaction solution is an intracellular solution, the cells into which the fluorescence-labeled siRNA is introduced can be placed in a fluorescence microscope having a confocal optical system, and a fluorescence signal can be detected by a conventional method (for example, (Refer nonpatent literature 2.). In addition, although the detection of the fluorescence signal analyzed by the single molecule fluorescence analysis method such as FCS generally uses a confocal optical system, the fluorescence derived from one molecule for performing the single molecule fluorescence analysis. The optical system is not particularly limited as long as it can acquire a signal, and an optical system 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.

  Examples of the single-molecule fluorescence analysis method used in the present invention include fluorescence correlation spectroscopy (FCS), fluorescence intensity distribution analysis method (Fluorescence-Intensity Distribution Analysis (FIDA)), and fluorescence polarization analysis method (FIDA-polarization: FIDA-PO). Etc.

  FCS is a technique that can determine the time (translational diffusion time) during which a fluorescently labeled molecule (fluorescently labeled molecule) in a reaction solution diffuses. The translational diffusion time depends on the size of the molecule. For example, the translational diffusion time for large molecules is longer than the translational diffusion time for small molecules. For this reason, FCS is a technique used to monitor changes in size due to molecular interaction, decomposition, aggregation, and the like. If there are fluorescently labeled molecules with different translational diffusion times (ie, molecules with different sizes) in the observation region, the number and ratio of molecules of each size can be calculated by performing a two-component analysis. It is also possible to calculate.

  FIG. 1 is a conceptual diagram showing the incorporation of siRNA having a fluorescent substance bound to the 5 'end of the sense strand into RISC. RISC (2) containing RNA helicase (2) against siRNA (1) composed of a sense strand (1s) having a fluorescent substance (F) bonded to the 5 ′ end and an antisense strand (1a) complementary thereto. When 3) acts from the 3 ′ end side of the sense strand (1s), the antisense strand (1a) is incorporated into RISC (3) and the sense strand (1s) is dissociated. Thereafter, mRNA (4) of the target gene having a base sequence complementary to the antisense strand (1a) is incorporated into RISC (3) and cleaved (on-target effect). The dissociated sense strand (1s) is decomposed by the action of exonuclease (6) and the like. On the other hand, when the RISC (3) containing the RNA helicase (2) acts on the siRNA (1) from the 5 ′ end side of the sense strand (1s), the sense strand (1s) becomes the RISC (3). And the antisense strand (1a) is dissociated. Thereafter, mRNA (5) of a gene other than the target gene having a base sequence complementary to the sense strand (1s) is taken into RISC (3) and cleaved (off-target effect). The dissociated antisense strand (1a) is degraded by the action of exonuclease (6) and the like.

  For this reason, when the method for evaluating the siRNA gene expression suppression effect of the present invention is performed using a fluorescently labeled siRNA in which a fluorescent substance is bound to the 5 ′ end of the sense strand, an on-target effect occurs, that is, antisense. When the strand is incorporated into RISC or when the RNA helicase is cleaved by acting from the 3 ′ end side of the sense strand, the sense strand dissociates into a single strand, so in step (b) The translational diffusion time (t2) obtained by the analysis in the step (c) is shorter than the translational diffusion time (t1) obtained by the analysis. Furthermore, when a single-stranded RNA-specific degrading enzyme or the like such as exonuclease is present in the reaction solution, the sense strand that is a free single-stranded RNA is degraded, resulting in the step (c). The translational diffusion time (t2) obtained by the analysis becomes shorter. Conversely, when an off-target effect occurs, that is, when the sense strand is incorporated into RISC, or when the RNA helicase is cleaved by acting from the 5 ′ end side of the sense strand, the sense strand is RNA Since it is combined with helicase or RISC, the translational diffusion time (t2) obtained by the analysis in the step (c) is longer than the translational diffusion time (t1) obtained by the analysis in the step (b).

  Therefore, in the step (d), when the translational diffusion time (t2) is shorter than the translational diffusion time (t1), the fluorescence-labeled siRNA is preferentially incorporated into the RISC, and the on-target It is evaluated that the effect is large and the gene expression suppression effect is high. Conversely, when the translational diffusion time (t2) is longer than the translational diffusion time (t1), the sense strand is preferentially taken into RISC, and it is evaluated that the off-target effect is large and the gene expression suppression effect is low. .

  When both an on-target effect and an off-target effect occur for a certain fluorescently labeled siRNA, these molecules having different translational diffusion times are mixed in the reaction solution. The percentage of molecules with each translational diffusion time can be calculated. For this reason, it is possible to calculate the uptake efficiency of siRNA into RISC. In the present invention, “siRNA incorporation efficiency into RISC” means “number of siRNA molecules in which on-target effect has occurred” / {[number of siRNA molecules in which on-target effect has occurred] + [off-target effect is Number of siRNA molecules generated]}.

  FIDA is a technique that can determine the fluorescence intensity per molecule and the number of fluorescently labeled molecules in a reaction solution. Since the fluorescence characteristics vary depending on the type of fluorescent substance, the fluorescence intensity per molecule may vary depending on the state of the bound nucleic acid. That is, fluorescently labeled RNA may have different fluorescence intensity per molecule depending on the base sequence and length, whether it is double-stranded RNA or single-stranded RNA, and the environmental state of the fluorescent substance.

  Therefore, by analyzing using FIDA, the fluorescence intensity (Q1) obtained by the analysis in step (b) (that is, the fluorescence intensity per molecule of the fluorescence-labeled siRNA that is a double-stranded RNA), and the step By comparing with the fluorescence intensity (Q2) obtained by the analysis in (c), is the fluorescently labeled molecule present in the reaction solution of step (c) in a state of single-stranded RNA by cleavage? It can be inferred whether it is in a state where it has become a further single-stranded RNA due to degradation, or in a state where it is bound to a protein such as an RNA helicase. For example, when TAMRA is used as the fluorescent substance, if the fluorescence intensity (Q2) is smaller than the fluorescence intensity (Q1), the fluorescently labeled molecule present in the reaction solution of step (c) is single-stranded by cleavage. It is considered that the RNA has been formed. Furthermore, when it is decomposed by exonuclease or the like, the fluorescence intensity per molecule becomes small.

  Therefore, when TAMRA is used as the fluorescent substance, in step (d), when the fluorescence intensity (Q2) is smaller than the fluorescence intensity (Q1), the fluorescence-labeled siRNA has priority to the antisense strand. Incorporated into RISC, it is evaluated that the on-target effect is large and the gene expression suppression effect is high. Conversely, when the fluorescence intensity (Q2) is higher than the fluorescence intensity (Q1), the sense strand is preferentially taken into RISC, and it is evaluated that the off-target effect is large and the gene expression suppression effect is low.

  Also in FIDA, when there are fluorescently labeled molecules with different fluorescence intensities (that is, molecules with different states) in the observation region, the number and ratio of molecules in each state are obtained by performing a two-component analysis. Can also be calculated. Therefore, even when analysis is performed using FIDA, it is possible to calculate the efficiency of siRNA incorporation into RISC as in FCS.

  FIDA-PO is a technique in which fluorescence intensity distribution and fluorescence polarization analysis are combined, and the degree of fluorescence polarization and the number of molecules of a fluorescently labeled molecule can be determined. The degree of fluorescence polarization varies depending on the size of the molecule. For example, a small molecule has a faster molecular rotational movement than a large molecule, and the degree of fluorescence polarization is small.

  For this reason, when the method for evaluating the siRNA gene expression suppression effect of the present invention is performed using a fluorescently labeled siRNA in which a fluorescent substance is bound to the 5 ′ end of the sense strand, an on-target effect occurs, that is, antisense. When the strand is incorporated into RISC or when the RNA helicase is cleaved by acting from the 3 ′ end side of the sense strand, the sense strand dissociates into a single strand, so in step (b) The fluorescence polarization degree (mP2) obtained by the analysis in the step (c) is smaller than the fluorescence polarization degree (mP1) obtained by the analysis. Furthermore, when a single-stranded RNA-specific degrading enzyme or the like such as exonuclease is present in the reaction solution, the sense strand that is a free single-stranded RNA is degraded, resulting in the step (c). The fluorescence polarization degree (mP2) obtained by the analysis becomes smaller. Conversely, when an off-target effect occurs, that is, when the sense strand is incorporated into RISC, or when the RNA helicase is cleaved by acting from the 5 ′ end side of the sense strand, the sense strand is RNA Since it is bound to helicase or RISC, the fluorescence polarization degree (mP2) obtained by the analysis in the step (c) is larger than the fluorescence polarization degree (mP1) obtained by the analysis in the step (b).

  Therefore, in the step (d), when the fluorescence polarization degree (mP2) is smaller than the fluorescence polarization degree (mP1), the fluorescence-labeled siRNA is preferentially incorporated into the RISC, and the on-target It is evaluated that the effect is large and the gene expression suppression effect is high. Conversely, when the degree of fluorescence polarization (mP2) is greater than the degree of fluorescence polarization (mP1), the sense strand is preferentially incorporated into RISC, and the off-target effect is large and the gene expression suppression effect is low. .

  Also in FIDA-PO, when there are fluorescently labeled molecules with different degrees of fluorescence polarization (that is, molecules with different sizes) in the observation region, by performing a two-component analysis, It is also possible to calculate the number and proportion of molecules. Therefore, even when analysis is performed using FIDA-PO, siRNA incorporation efficiency into RISC can be calculated in the same manner as FCS.

  When an intracellular solution is used as the reaction solution in the step (b) or (c), that is, when a fluorescent signal is detected by introducing a fluorescently labeled siRNA into a cell, the parameter of the molecular diffusion movement is included in the analysis. By using FIDA or FIDA-PO which is not included, interpretation of data becomes easy. This is because the cells are not a uniform medium, but are filled with organelles such as cytoskeleton, endoplasmic reticulum, mitochondria, etc., and the fluorescently labeled molecules in the cells do not have a uniform diffusion movement. In FCS, molecular diffusion is observed. Since this is based on the premise of free diffusion, data analysis is complicated when analyzing fluorescent signals detected from fluorescent molecules in cells by FCS. There is a case.

  When the siRNA to be evaluated is evaluated as having a low gene expression suppression effect in step (d), siRNA design is performed again to synthesize a new siRNA, and this is similarly performed in steps (a) to (a) to (c). (D) can be performed and the gene expression suppression effect can be evaluated. By repeating this operation, an optimal siRNA for performing RNA interference of the target gene can be obtained.

  In addition, when there are two or more types of siRNAs to be evaluated, the above steps (a) to (d) are performed on each siRNA, and the gene expression suppression effect of each siRNA is evaluated. The siRNA having the highest evaluation can be selected as the most suitable siRNA for performing RNA interference of the target gene.

  By using the siRNA gene expression inhibitory effect evaluation method of the present invention, mRNA can be extracted from cells into which siRNA has been introduced, and the siRNA gene expression inhibitory effect can be rapidly achieved without work such as reverse transcription and real-time PCR. Can be evaluated. In particular, when the step (b) or (c) is performed in vitro using an extracellular solution as a reaction solution, a complicated cell culture operation or the like is not required, and a reagent such as siRNA is simply added and mixed. In addition, since the fluorescence signal can be detected and analyzed, the evaluation can be performed quickly even when there are a large number of siRNAs to be evaluated.

  By using the siRNA gene expression inhibitory effect evaluation method of the present invention, the off-target effect can be confirmed without using cells, so that the optimal siRNA can be selected from a plurality of candidate siRNAs as candidates. It is possible to improve the design accuracy of siRNA simply. Moreover, also when performing a process (b) or (c) in a cell, it becomes possible to simply confirm the uptake | capture to RISC, without disrupting a cell, and RNA interference can be performed more easily.

  In addition, the case where the step (b) or (c) is performed in vitro is simpler than the case where the step (b) or (c) is performed in vivo, and an evaluation from which the influence of proteins other than RISC is removed can be obtained. Therefore, in the case of evaluating one siRNA, the step (b) or (c) is performed in vitro, and the step (b) or (c) is performed only for siRNA evaluated to have a high gene expression suppression effect. It can also be performed in vivo and evaluated by the method for evaluating the effect of suppressing gene expression of siRNA of the present invention.

  In the present invention, the gene expression suppression effect is evaluated for the step of incorporating an antisense strand into RISC, and the subsequent steps are not evaluated. For this reason, for siRNA evaluated to have a high gene expression suppression effect in the present invention, mRNA is extracted from the cells into which the siRNA has been introduced by a conventional method, and the target is obtained by a conventional method such as reverse transcription reaction or real-time PCR. It is also preferable to confirm that the amount of mRNA of the gene is reduced.

  FIG. 2 shows one embodiment of the method for evaluating the siRNA gene expression inhibitory effect of the present invention when an extracellular solution is used as a reaction solution using a fluorescently labeled siRNA in which the fluorescent substance TAMRA is bound to the 5 ′ end of the sense strand. It is the flowchart figure which showed. In addition, it cannot be overemphasized that the gene expression suppression effect evaluation method of siRNA of this invention is not limited to this aspect. First, as step (a), a base sequence of fluorescently labeled siRNA (TAMRA-siRNA) in which TAMRA is bound to the 5 ′ end of the sense strand to be evaluated for gene expression suppression effect is designed (step 01). Synthesize. Next, as step (b), a reaction solution containing TAMRA-siRNA is prepared (step 02), the reaction solution is irradiated with TAMRA excitation light, a fluorescence signal is detected, and a single molecule fluorescence analysis method is performed. (Steps 03-1 to 3). The translational diffusion time (t1) when analyzed using FCS, the fluorescence intensity (Q1) when analyzed using FIDA, and the degree of fluorescence polarization (mP1) when analyzed using FIDA-PO Are calculated respectively. Next, as step (c), TAMRA-siRNA is added to the reaction solution containing RISC and ATP and reacted (step 04), and the reaction solution is irradiated with TAMRA excitation light. A fluorescence signal is detected and analyzed by single molecule fluorescence analysis (steps 05-1 to 3). The translational diffusion time (t2) when analyzed using FCS, the fluorescence intensity (Q2) when analyzed using FIDA, and the degree of fluorescence polarization (mP2) when analyzed using FIDA-PO Are calculated respectively. Furthermore, as a process (d), the gene expression suppression effect of TAMRA-siRNA is evaluated (steps 06-1 to 3). When the translational diffusion time (t2) is longer than the translational diffusion time (t1), when the fluorescence intensity (Q2) is greater than the fluorescence intensity (Q1), the fluorescence polarization degree (mP2) is greater than the fluorescence polarization degree (mP1). If it is larger, it is evaluated that the gene expression suppression effect is low, siRNA is redesigned (step 01), and step 02 to step 06 are repeated. On the other hand, when the translational diffusion time (t2) is shorter than the translational diffusion time (t1), when the fluorescence intensity (Q2) is smaller than the fluorescence intensity (Q1), the fluorescence polarization degree (mP1) If mP2) is smaller, it is evaluated that the effect of suppressing gene expression is high, a two-component analysis is performed (step 07), and siRNA incorporation efficiency (reaction efficiency) into RISC is determined (step 08). About siRNA with favorable reaction efficiency, it can also introduce | transduce into a cultured cell and can also confirm the gene expression suppression effect in more physiological conditions.

  FIG. 3 shows an embodiment of the method for evaluating the siRNA gene expression inhibitory effect of the present invention when a fluorescent solution TAMRA is bound to the 5 ′ end of the sense strand and the intracellular solution is used as a reaction solution. It is the flowchart figure which showed. In addition, it cannot be overemphasized that the gene expression suppression effect evaluation method of siRNA of this invention is not limited to this aspect. First, as step (a), a base sequence of fluorescently labeled siRNA (TAMRA-siRNA) in which TAMRA is bound to the 5 ′ end of the sense strand to be evaluated for gene expression suppression effect is designed (step 101). Synthesize. Next, as step (b), TAMRA-siRNA is introduced into RISC knockdown cells (step 102), the cells are cultured (step 103), the cells are observed, and the state of introduction is confirmed (step 104). . Instead of steps 102 to 104, a reaction solution containing TAMRA-siRNA may be prepared using an extracellular solution (step 105). A TAMRA-siRNA-introduced cell or an extracellular solution containing TAMRA-siRNA is irradiated with TAMRA excitation light to detect a fluorescent signal and analyzed by single molecule fluorescence analysis (steps 106-1 to 106-3). ). The translational diffusion time (t1) when analyzed using FCS, the fluorescence intensity (Q1) when analyzed using FIDA, and the degree of fluorescence polarization (mP1) when analyzed using FIDA-PO Are calculated respectively. Next, as step (c), TAMRA-siRNA is introduced into cells expressing RISC (step 107), the cells are observed, and the state of introduction is confirmed (step 108). A TAMRA-siRNA-introduced cell is irradiated with TAMRA excitation light, a fluorescent signal is detected, and analyzed by a single molecule fluorescence analysis method (steps 109-1 to 109). The translational diffusion time (t2) when analyzed using FCS, the fluorescence intensity (Q2) when analyzed using FIDA, and the degree of fluorescence polarization (mP2) when analyzed using FIDA-PO Are calculated respectively. Furthermore, as a process (d), the gene expression suppression effect of TAMRA-siRNA is evaluated (steps 110-1 to 3). When the translational diffusion time (t2) is longer than the translational diffusion time (t1), when the fluorescence intensity (Q2) is greater than the fluorescence intensity (Q1), the fluorescence polarization degree (mP2) is greater than the fluorescence polarization degree (mP1). If the value is larger, it is evaluated that the gene expression suppression effect is low, siRNA is redesigned again (step 101), and step 102 to step 109 are repeated. On the other hand, when the translational diffusion time (t2) is shorter than the translational diffusion time (t1), when the fluorescence intensity (Q2) is smaller than the fluorescence intensity (Q1), the fluorescence polarization degree (mP1) When mP2) is smaller, it is evaluated that the gene expression suppression effect is high. Thereafter, the mRNA of the cell may be further extracted, and it may be confirmed that the amount of mRNA of the target gene is actually decreasing (step 111).

As described above, it is often difficult to obtain cells that do not express RNA helicase or RISC. On the other hand, in single-molecule fluorescence analysis, the behavior of fluorescently labeled molecules in cells in a state where RNA interference does not occur is approximately equal between double-stranded RNAs labeled with the same type of fluorescent substance.
Therefore, instead of step (b), an RNA helicase or the like is expressed by using an siRNA that is not unwound or cleaved by an RNA helicase and is not incorporated into RISC and is labeled with the same type of fluorescent substance. The fluorescence signal of the fluorescence-labeled siRNA in the cell before being affected by RISC or the like (before RNA interference) can be detected and analyzed using the living cells.

Specifically, it is a method for evaluating the effect of suppressing gene expression by siRNA, characterized by comprising the following steps (a ′) to (d ′).
(A ′) A step of preparing a fluorescently labeled siRNA in which either one of the sense strand or the antisense strand of the siRNA to be evaluated is labeled with a fluorescent substance.
(B ′) siRNA that does not undergo unwinding of the RNA helicase labeled with the fluorescent substance, or siRNA that is not incorporated into the RISC labeled with the fluorescent substance is introduced into the cell, A step of irradiating light having a wavelength capable of exciting a fluorescent substance, detecting fluorescence generated from the fluorescent substance as a fluorescent signal, and analyzing the detected fluorescent signal by a single molecule fluorescence analysis method.
(C ′) After introducing and reacting the fluorescently labeled siRNA into a cell, the cell is irradiated with light having a wavelength capable of exciting the fluorescent substance, and fluorescence generated from the fluorescent substance is used as a fluorescent signal. A step of detecting and analyzing the detected fluorescence signal by single molecule fluorescence analysis.
(D ′) A step of comparing the analysis result obtained in the step (b ′) with the analysis result obtained in the step (c ′) to evaluate the gene expression suppression effect of the siRNA.

Here, step (a ′) is the same as step (a) described above.
In the step (b ′), in place of the fluorescence-labeled siRNA that is the evaluation target in the above-mentioned step (b), siRNA that does not undergo unwinding of the RNA helicase or siRNA that is not incorporated into the RISC (negative control siRNA) This is the same as the case where the reaction solution is an intracellular solution, except that siRNA fluorescently labeled with the same type of fluorescent substance as that of siRNA is used.

  By using siRNA that does not undergo unwinding by RNA helicase or siRNA in which single-stranded RNA that has been unwound and cleaved by RNA helicase is not taken into RISC as a negative control siRNA, the same cells as in step (c ′) are used. Even when it is used, the behavior of fluorescently labeled siRNA in cells before being affected by RISC or the like can be observed. As such a negative control siRNA, for example, siRNA having a covalent bond between strands can be used.

  The base pair length of the negative control siRNA is not particularly limited, but is preferably about the same as the base pair length of the siRNA to be evaluated (about ± 5 bp), more preferably the same base pair length. preferable. When the base pair length is about the same, the fluorescence labeling molecule in which the behavior of the fluorescently labeled molecule in the cell of the negative control siRNA does not cause more RNA interference is compared to the case where the difference is greatly different. This is to make it more approximate to siRNA.

Step (c ′) is the same as in the above-described step (c) when the reaction solution is an intracellular solution.
Further, in the evaluation in the step (d ′), the analysis result obtained in the step (b ′) and the analysis result obtained in the step (c ′) are compared in the same manner as in the step (d). The effect of suppressing the gene expression of the fluorescently labeled siRNA to be evaluated can be evaluated.

  When the siRNA to be evaluated is evaluated as having a low gene expression suppression effect in the step (d ′), the siRNA design is performed again to synthesize a new siRNA, and this is similarly performed in the step (a ′ ) To (d ′), and the gene expression inhibitory effect can be evaluated. By repeating this operation, an optimal siRNA for performing RNA interference of the target gene can be obtained.

  Moreover, when there are two or more types of siRNAs to be evaluated, the steps (a ′) to (d ′) are performed on each siRNA, and the gene expression suppression effect of each siRNA is evaluated. SiRNA having the highest evaluation can be selected as the most suitable siRNA for performing RNA interference of the target gene.

  In the same manner as described above, after introducing the positive control siRNA into the cells into which siRNA is introduced in steps (b ′) and (c ′) and reacting, the same as in step (c), You may analyze the fluorescence signal detected by irradiating the light of a suitable wavelength by the single molecule fluorescence analysis method.

  FIG. 4 includes steps (a ′) to (d ′) in the case of using a fluorescently labeled siRNA in which the fluorescent substance TAMRA is bound to the 5 ′ end of the sense strand and a negative control siRNA in which TAMRA is also bound. It is the flowchart figure which showed the one aspect | mode of the siRNA gene expression inhibitory effect evaluation method of this invention. In addition, it cannot be overemphasized that the gene expression suppression effect evaluation method of siRNA of this invention is not limited to this aspect. First, as step (a ′), a nucleotide sequence of fluorescently labeled siRNA (TAMRA-siRNA) in which TAMRA is bound to the 5 ′ end of the sense strand to be evaluated for gene expression suppression effect is designed (step 201). Is synthesized. Next, as step (b ′), a negative control siRNA conjugated with TAMRA (control TAMRA-labeled siRNA) is introduced into the cells (step 202), the cells are cultured (step 203), and the cells are observed. The state of introduction is confirmed (step 204). The cells into which the control TAMRA-labeled siRNA has been introduced are irradiated with TAMRA excitation light to detect a fluorescent signal and analyzed by single-molecule fluorescence analysis (steps 206-1 to 206-3). The translational diffusion time (t1) when analyzed using FCS, the fluorescence intensity (Q1) when analyzed using FIDA, and the degree of fluorescence polarization (mP1) when analyzed using FIDA-PO Are calculated respectively. Next, as step (c ′), TAMRA-siRNA is introduced into the same type of cells used in step 202 (step 207), the cells are observed, and the state of introduction is confirmed (step 208). A TAMRA-siRNA-introduced cell is irradiated with TAMRA excitation light, a fluorescent signal is detected, and analyzed by single molecule fluorescence analysis (steps 209-1 to 3). The translational diffusion time (t2) when analyzed using FCS, the fluorescence intensity (Q2) when analyzed using FIDA, and the degree of fluorescence polarization (mP2) when analyzed using FIDA-PO Are calculated respectively. Further, as a step (d ′), the gene expression inhibitory effect of TAMRA-siRNA is evaluated (steps 210-1 to 210-3). When the translational diffusion time (t2) is longer than the translational diffusion time (t1), when the fluorescence intensity (Q2) is greater than the fluorescence intensity (Q1), the fluorescence polarization degree (mP2) is greater than the fluorescence polarization degree (mP1). If it is larger, it is evaluated that the gene expression suppression effect is low, siRNA is redesigned again (step 201), and step 202 to step 209 are repeated. On the other hand, when the translational diffusion time (t2) is shorter than the translational diffusion time (t1), when the fluorescence intensity (Q2) is smaller than the fluorescence intensity (Q1), the fluorescence polarization degree (mP1) When mP2) is smaller, it is evaluated that the gene expression suppression effect is high. Thereafter, the mRNA of the cell may be further extracted, and it may be confirmed that the amount of mRNA of the target gene is actually decreasing (step 211).

  In addition, in this invention, the gene expression suppression effect of siRNA can also be evaluated by performing FCCS as a single molecule fluorescence analysis method. For example, a fluorescently labeled siRNA is introduced into a cell expressing RISC (fluorescently labeled RISC cell) labeled with a fluorescent material having a spectral characteristic different from the fluorescent material used for labeling the siRNA to be evaluated, and each fluorescence Two kinds of wavelengths of light that can excite each substance are irradiated, and fluorescence generated from each fluorescent substance is detected as a fluorescence signal and analyzed by FCCS. Since it can be determined whether or not the fluorescence-labeled RISC and the fluorescence-labeled siRNA interact with each other by FCCS analysis, the effect of suppressing siRNA gene expression can be evaluated as in the case of using FCS or the like. As the fluorescence-labeled RISC cell, for example, a cell expressing a chimeric protein obtained by modifying Ago, which is a constituent protein of RISC, with a fluorescent protein such as GFP can be used (see, for example, Non-Patent Document 2).

  By detecting the fluorescence signal over time and measuring the cross-correlation change by FCCS measurement, the interaction between the fluorescently labeled RISC and the fluorescently labeled siRNA can be measured in more detail. For example, when there is a cross-correlation immediately after introducing a fluorescently labeled siRNA labeled with a sense strand or at a relatively early stage, and the cross-correlation disappears over time, the fluorescently labeled siRNA interacts with the fluorescently labeled RISC. It can be evaluated that the antisense strand produced by cleavage is then incorporated into RISC and the sense strand is dissociated (on-target effect), and the siRNA gene expression suppression effect is high. . On the other hand, if there is a cross-correlation immediately after introduction or at a relatively early time, and there is a cross-correlation even when the subsequent time has elapsed, the sense strand generated by cleavage is incorporated into the RISC (off-target effect) Therefore, it can be evaluated that the siRNA has a low gene expression inhibitory effect.

  In the present invention, the state of the fluorescence-labeled siRNA before and after being affected by RISC or the like (before RNA interference) and after being affected by RISC or the like is observed in separate reaction solutions. The siRNA gene expression inhibitory effect is evaluated by comparing the fluorescence signal siRNA over time immediately after adding the fluorescently labeled siRNA to be evaluated to the reaction solution containing RNA helicase or RISC and ATP. By detecting single-molecule fluorescence and performing single-molecule fluorescence analysis, it is also possible to evaluate the gene expression suppression effect of siRNA only from the temporal change of the fluorescence-labeled siRNA in the reaction solution. The state of the labeled siRNA immediately after addition to the reaction solution corresponds to the state before being affected by RISC or the like (before RNA interference), and the analysis result immediately after the addition is the analysis result obtained in the above-described step (b). It is because it corresponds to. In this method, similarly to the reaction solution in the above-mentioned step (c), the reaction may be performed in an extracellular solution, or may be performed in a cell after introducing fluorescently labeled siRNA into the cell.

  EXAMPLES Next, although an Example is shown and this invention is demonstrated further in detail, this invention is not limited to a following example.

[Example 1]
<Preparation of RNA helicase>
Of the cDNA sequence of FLJ10383 of ATP-dependent RNA helicase (TOYOBO, NCBI accession No: K001245), a partial region having a total length of 1-1362 is incorporated into the multicloning site of the protein expression vector pET21a (manufactured by Novagen) in E. coli. An RNA helicase expression vector (pET21a-RNAhelicase) was constructed.
This pET21a-RNAhelicase was introduced into Escherichia coli BL21 (DE3) strain (manufactured by Novagen) and cultured overnight to obtain a culture solution containing E. coli expressing RNA helicase. Thereafter, the E. coli recovered from the culture solution is added with bugbuster protein extraction reagent (manufactured by Novagen) to disrupt the E. coli, solubilize the protein, add a protease inhibitor, and treat at 24 ° C. for 20 minutes. As a result, a cell extract was obtained. The RNA helicase was purified from the cell extract by performing His-Tag purification using His-Spintrap (manufactured by GE Healthcare). For elution of RNA helicase from the His-Spintrap column, Imidazole Solution was used. By confirming the band by SDS-PAGE (molecular weight 48647 Da, about 48 KDa), it was confirmed that the expressed RNA helicase was purified.

<Reaction with fluorescently labeled siRNA>
As the fluorescently labeled siRNA, FAM-GAPDH siRNA positive control (FAM-siRNA) (Ambion) was used. This FAM-siRNA is commercially available as a positive control in RNA interference, and it is considered that the off-target effect is small. In FAM-siRNA, FAM is bound to the 5 ′ end of the sense strand.
Using this FAM-siRNA and the RNA helicase prepared above, the effect of suppressing the GAPDH gene expression of FAM-siRNA was evaluated. The reaction using RNA helicase was carried out with reference to the method of Huang et al. (Journal of Biological Chemistry, 2002, 277, 12810).
First, FAM-siRNA, ATP (A3377) was added to a buffer (70 mM Tris-HCl, 200 mM NaCl, 1 mM MgCl ₂ , 5 mM DTT, 40 units RNasin (manufactured by N2111 Promega)) so that the final concentration was as shown in Table 1. Sigma) and RNA helicase were added to prepare four types of reaction solutions containing FAM-siRNA (samples 1 to 4; final volume 20 μl). In the table, the concentration of RNA helicase means the concentration per total protein of the purified RNA helicase prepared above.

  After the prepared samples 1 to 4 were reacted by incubating at 37 ° C. for 60 minutes, using a single molecule fluorescence analyzer MF20 (manufactured by Olympus), these samples were subjected to 488 nm laser light (laser power: 100 μW) and a fluorescent signal was detected. The measurement method was measured 5 times for 15 seconds per sample using FCS or FIDA.

  FIG. 5A is a diagram showing the translational diffusion time (μ seconds) obtained from each sample by FCS analysis. 5 (B) shows the state of FAM-siRNA in sample 1, FIG. 5 (C) shows the state of FAM-siRNA in sample 2, and FIG. 5 (D) shows the FAM-siRNA in samples 3 and 4. It is the figure which showed the state of siRNA typically.

  Sample 1 is the translational diffusion time of FAM-siRNA, which is a double-stranded RNA before being affected by RNA helicase. In sample 2 where the ATP concentration was 0 nM, the translational diffusion time was longer than in sample 1. This is presumably because the RNA helicase in Sample 2 bound to FAM-siRNA, resulting in an increase in size per molecule. On the other hand, in Samples 3 and 4 to which ATP was added, the translational diffusion time was reduced as compared with Sample 2. This is presumably because the RNA helicase was activated by ATP, and the double-stranded FAM-siRNA was unwound and exists as a single-stranded RNA having a lower molecular weight. In Sample 4 to which 16 mM ATP was added, the translational diffusion time was longer than that in Sample 1, but this was because RISC was not present in the sample, so that the RNA helicase was labeled with a FAM-labeled sense strand. There is a mixture of unbound antisense strands, so the translational diffusion time is on average longer than when only the FAM-labeled sense strand is present. It is thought that it has become.

  FIG. 6A shows the fluorescence intensity Q (kHz) obtained from each sample by FIDA analysis. 6 (B) shows the state of FAM-siRNA in sample 1, FIG. 6 (C) shows the state of FAM-siRNA in sample 2, and FIG. 6 (D) shows the FAM-siRNA in samples 3 and 4. It is the figure which showed the state of siRNA typically.

  Similar to FCS, sample 1 is the fluorescence intensity of FAM-siRNA, which is a double-stranded RNA before being affected by RNA helicase. In sample 2 where the ATP concentration was 0 nM, the fluorescence intensity increased compared to sample 1. This is presumed that the RNA helicase in sample 2 bound to FAM-siRNA, so that the environment around FAM became hydrophobic and the fluorescence intensity increased. On the other hand, in samples 3 and 4 to which ATP was added, the fluorescence intensity was decreased as compared with sample 2. This is because the RNA helicase is activated by ATP, the double-stranded FAM-siRNA is unwound, and the RNA helicase binds to the antisense strand that is not labeled more than the FAM-labeled sense strand. It is assumed that the environment surrounding the FAM has become hydrophilic and the fluorescence intensity has decreased.

  By using the siRNA gene expression inhibitory effect evaluation method of the present invention, the gene expression inhibitory effect of siRNA used in RNA interference can be evaluated quickly and easily. It is possible to simply screen highly effective siRNA, and it can be used not only in the field of academic research but also in the field of development of nucleic acid drugs using siRNA.

It is the conceptual diagram which showed the uptake | capture to the RISC of siRNA which combined the fluorescent substance with 5 'terminal of the sense strand. It is the flowchart figure which showed the one aspect | mode of the gene expression suppression effect evaluation method of siRNA of this invention at the time of making an extracellular solution into a reaction solution. It is the flowchart figure which showed the one aspect | mode of the gene expression suppression effect evaluation method of siRNA of this invention at the time of making intracellular solution into a reaction solution. The siRNA of the present invention having the steps (a ′) to (d ′) in the case of using a fluorescently labeled siRNA in which the fluorescent substance TAMRA is bound to the 5 ′ end of the sense strand and a negative control siRNA in which TAMRA is also bound. It is the flowchart figure which showed the one aspect | mode of the gene expression suppression effect evaluation method. FIG. 5A is a diagram showing the translational diffusion time (μ seconds) obtained from each sample by FCS analysis in Example 1. FIG. 5 (B) shows the state of FAM-siRNA in sample 1, FIG. 5 (C) shows the state of FAM-siRNA in sample 2, and FIG. 5 (D) shows the FAM-siRNA in samples 3 and 4. It is the figure which showed the state of siRNA typically. FIG. 6A is a diagram showing the fluorescence intensity Q (kHz) obtained from each sample by FIDA analysis in Example 1. FIG. 6 (B) shows the state of FAM-siRNA in sample 1, FIG. 6 (C) shows the state of FAM-siRNA in sample 2, and FIG. 6 (D) shows the FAM-siRNA in samples 3 and 4. It is the figure which showed the state of siRNA typically.

Explanation of symbols

  F ... fluorescent substance, 1 ... siRNA, 1s ... sense strand, 1a ... antisense strand, 2 ... RNA helicase, 3 ... RISC, 4 ... mRNA of target gene, 5 ... mRNA of gene other than target gene, 6 ... exonuclease .

Claims (5)

A method for evaluating the effect of suppressing gene expression by siRNA,
(A) preparing a fluorescently labeled siRNA in which either one of the sense strand or the antisense strand of the siRNA to be evaluated is labeled with a fluorescent substance;
(B) The reaction solution containing the fluorescently labeled siRNA 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, and the detected fluorescent signal is single-molecule fluorescent. A process of analyzing by an analysis method;
(C) After reacting the reaction solution containing RNA helicase or RISC and ATP by adding the fluorescent-labeled siRNA, the reaction solution is irradiated with light having a wavelength capable of exciting the fluorescent substance, and Detecting fluorescence generated from a fluorescent substance as a fluorescence signal, and analyzing the detected fluorescence signal by a single molecule fluorescence analysis method;
(D) comparing the analysis result obtained in the step (b) with the analysis result obtained in the step (c), and evaluating the gene expression inhibitory effect of the siRNA;
A method for evaluating the effect of suppressing gene expression of siRNA, comprising:   The siRNA gene expression inhibitory effect evaluation method according to claim 1, wherein there are two or more types of siRNA to be evaluated, and the steps (a) to (d) are performed on each siRNA. A method for evaluating the effect of suppressing gene expression by siRNA,
(A ′) preparing a fluorescently labeled siRNA in which either one of the sense strand or the antisense strand of the siRNA to be evaluated is labeled with a fluorescent substance;
(B ′) siRNA that does not undergo unwinding of the RNA helicase labeled with the fluorescent substance, or siRNA that is not incorporated into the RISC labeled with the fluorescent substance is introduced into the cell, Irradiating light having a wavelength capable of exciting the fluorescent substance, detecting fluorescence generated from the fluorescent substance as a fluorescent signal, and analyzing the detected fluorescent signal by a single molecule fluorescence analysis method;
(C ′) After introducing and reacting the fluorescently labeled siRNA into a cell, the cell is irradiated with light having a wavelength capable of exciting the fluorescent substance, and fluorescence generated from the fluorescent substance is used as a fluorescent signal. Detecting and analyzing the detected fluorescence signal by single molecule fluorescence analysis,
(D ′) comparing the analysis result obtained in the step (b ′) with the analysis result obtained in the step (c ′) to evaluate the gene expression inhibitory effect of the siRNA;
A method for evaluating the effect of suppressing gene expression of siRNA, comprising:   The method for evaluating the siRNA gene expression suppression effect according to any one of claims 1 to 3, wherein the fluorescently labeled siRNA has a sense strand labeled with a fluorescent substance.   The single-molecule fluorescence analysis methods include fluorescence correlation spectroscopy (FCS), fluorescence intensity distribution analysis method (Fluorescence-Intensity Distribution Analysis: FIDA), and fluorescence polarization analysis method (FIDA polarization-ID: FIDA). The method for evaluating the siRNA gene expression suppression effect according to any one of claims 1 to 4, wherein the method is selected from the group consisting of:

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