JP2011036132A - Method for measuring winding-back reaction efficiency of helicase - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for precisely measuring and evaluating the enzymatic activity of helicase. <P>SOLUTION: The method for measuring the winding-back reaction efficiency of helicase includes (a) a step of adding and reacting the helicase to/with a fluorescent labeled double-strand nucleic acid comprising first fluorescent labeled single strand nucleic acid and second fluorescent labeled single strand nucleic acid and then measuring the apparent content of the fluorescent labeled double-strand nucleic acid in the reaction solution by a one molecular fluorescent analysis method, (b) a step of preparing the calibration curve sample series of calibration-curve fluorescent-labeled double strand nucleic acid labeled by the first fluorescent substance and the second fluorescent substance, (c) a step of measuring the apparent content of the calibration-curve fluorescent-labeled double strand nucleic acid in each calibration curve sample in the same way as in the step (a), (d) a step of making an approximate line representing the relationship between the apparent content measured in the step (c) and the real content in the measured sample, and (e) a step of calculating the winding-back reaction efficiency of the helicase on the basis of the approximate line made in the step (d) and the apparent content measured in the step (a). <P>COPYRIGHT: (C)2011,JPO&INPIT

Description

  The present invention relates to a method for accurately measuring the unwinding reaction efficiency of a helicase with respect to a double-stranded nucleic acid using a single molecule fluorescence analysis method.

  Helicase is a general term for enzymes that rewind a double helix of a double-stranded nucleic acid of DNA or RNA. It has been reported that helicases are involved in various actions involving genetic information such as DNA repair, replication, transcription, translation, and splicing, and it is understood that various helicases exist due to each action. Yes. Some helicases have also been found to cause abnormal diseases, and therefore helicase inhibitors are being developed.

  In this way, helicase is a diverse enzyme and plays an important role in vivo. However, details of substrate specificity, unwinding reaction efficiency, mechanism of action, etc. are still unclear. Many. Moreover, although the possibility of having helicase activity is suggested from the base sequence information, there are many unidentified enzymes whose actual activity has not been confirmed.

  In order to identify these helicases and elucidate the mechanism of action, it is important to measure the helicase activity. As a method for measuring the activity of helicase in vitro, for example, a double-stranded DNA labeled with a radioisotope and a helicase are mixed and reacted, and then a nucleobase in a sample (reaction solution) is electrophoresed. There is a method for performing quantification by separating a single-stranded nucleic acid and a double-stranded nucleic acid and detecting the amount of radioactivity. However, since this method uses a radioisotope, there is a problem in terms of simplicity, such as safety problems and the need for special facilities. In addition, since electrophoresis is further performed on a sample reacted with helicase, it takes time to detect.

  As a method for performing helicase activity more safely and simply, a measurement method using a fluorescent substance has been developed. For example, (1) a double-stranded nucleic acid having a 5 ′ protruding end, a double-stranded nucleic acid having a 3 ′ protruding end, and a double-stranded nucleic acid having no protruding end are each a single-stranded nucleic acid of a double-stranded nucleic acid. Prepared from a signal generating substance whose signal intensity is increased or decreased by dissociation into a double-stranded nucleic acid, subjected to an enzymatic reaction with a test helicase using the double-stranded nucleic acid as a substrate, and from a double-stranded nucleic acid to a single-stranded nucleic acid A method for identifying the substrate specificity of the test helicase by calculating the reaction rate of the test helicase to the nucleobase based on the change in signal intensity measured by the rewinding to is disclosed (for example, see Patent Document 1). .) In this method, a fluorescence resonance energy transfer (FRET) phenomenon or an intercalator using a fluorescent substance is used as a signal whose signal intensity increases or decreases by dissociation of a double-stranded nucleic acid into a single-stranded nucleic acid. ing.

  (2) a single-stranded nucleic acid having a fluorescent dye whose fluorescence intensity changes depending on proximity to or away from the base of the nucleic acid, and the other single-stranded nucleic acid having a base sequence complementary to the single-stranded nucleic acid; A step of preparing a double-stranded nucleic acid comprising: a step of carrying out an enzymatic reaction with helicase using the double-stranded nucleic acid as a substrate; and a step of measuring the fluorescence intensity from the reaction product of the enzymatic reaction. A measuring method is also disclosed (for example, see Patent Document 2). In this method, when a double-stranded nucleic acid of one of the double-stranded nucleic acids becomes a double-stranded nucleic acid, the site where the guanine base in the other nucleic acid strand is close is preliminarily fluorescent by the proximity of the guanine base. It is labeled with a fluorescent substance having the property of being quenched. The activity of helicase is detected by utilizing the fact that the fluorescence signal changes when this fluorescently labeled double-stranded nucleic acid undergoes unwinding by helicase to become a single-stranded nucleic acid.

  In addition, (3) the step of fluorescently labeling the nucleobase or helicase, the step of mixing the nucleobase and the helicase to form a mixed solution, the size of the substance having the fluorescent label in the mixed solution by the fluorescence analysis method, There is also disclosed a method for detecting a reaction between a nucleobase and a helicase, which comprises a step of obtaining brightness or number (see, for example, Patent Document 3). In this method, fluorescence correlation spectroscopy (Fluorescence Correlation Spectroscopy: FCS), fluorescence cross correlation spectroscopy (FCCS), fluorescence intensity distribution analysis method (Fluorescence Intensity Dimension ID) Item Fluorescence Intensity Multiplex Distribution Analysis (FIMDA) or Fluorescence Polarization Analysis (FIDA-PO) is mentioned.

JP 2005-253364 A JP 2008-99619 A JP 2005-80535 A

  In order to measure the efficiency of unwinding reaction by helicase, the ratio of double-stranded nucleic acid that has been unwound by helicase (one that has become single-stranded nucleic acid) and double-stranded nucleic acid that has not been unwound It is important to measure. Then, by measuring the unwinding reaction efficiency of the helicase, the reaction conditions such as the substrate specificity, optimal temperature, and optimal pH of the helicase can be examined in more detail. Furthermore, by measuring the unwinding reaction efficiency quantitatively, it is possible to compare the strength of activity with other helicases.

  However, in the above method (1), when FRET is used, since the fluorescence signal of FRET is weak in the state of double-stranded nucleic acid, double-stranded nucleic acid and single-stranded nucleic acid are distinguished and quantified. Is difficult. For this reason, depending on the method, it is possible to qualitatively determine the presence or absence of helicase activity, but it has been difficult to measure the efficiency of nucleobase unwinding reaction of helicase. In addition, when an intercalator is used, since a signal is not emitted from a single-stranded nucleic acid in the first place, the double-stranded nucleic acid cannot be quantified separately from the single-stranded nucleic acid.

  In the method (2), since the fluorescence of the entire measurement sample is detected, how much double-stranded nucleic acid is actually unwound by helicase in the sample being measured, That is, it was difficult to distinguish and quantify single-stranded nucleic acid and double-stranded nucleic acid. Therefore, even in this method, it was difficult to determine the efficiency of helicase activity.

On the other hand, in the method (3) above, the reaction between the fluorescently labeled double-stranded nucleic acid and the helicase is detected using a single-molecule fluorescence analysis method such as FCS. It is possible to distinguish and quantify. However, when helicase activity is measured by this method using FCS, in order to improve the discrimination accuracy between double-stranded nucleic acid and single-stranded nucleic acid, an enzyme that divides the single-stranded nucleic acid by exonuclease and reduces the molecular weight. When treatment is performed and such enzyme treatment is not performed, there is a problem that the accuracy of measuring the unwinding reaction efficiency of the helicase by this method is not always sufficient. Actually, in Patent Document 3, the unwinding reaction efficiency of helicase is not measured.
Patent Document 3 also describes detection using FCCS. However, it is essential for FCCS to use two types of molecules to be measured for the presence or absence of binding with fluorescent substances having different spectral characteristics, and in order to measure these two types of fluorescently labeled molecules, The sample must be irradiated with light of two different wavelengths using the same objective lens, and there is a problem that the irradiation area of the sample is shifted due to chromatic aberration of the objective lens. Another problem is that the properties of the fluorescent substances that label each molecule are different. For this reason, in FCCS, even when molecules labeled with two different types of fluorescent substances are bound to 100%, the binding efficiency of the obtained data is only much less than 100%. There is a principle problem that it is not detected. For this reason, when the helicase activity was measured by the above method (3) using FCCS, only qualitative findings to the extent of confirming the presence or absence of helicase activity were obtained.

  The present invention provides a method for accurately measuring and evaluating a more detailed helicase enzyme activity such as substrate specificity and reaction efficiency with respect to a helicase reaction that unwinds a double-stranded nucleic acid into a single-stranded nucleic acid. Objective.

  As a result of intensive studies to solve the above-mentioned problems, the present inventor previously measured the actual 2 in the measurement sample when measuring the reaction between the fluorescently labeled double-stranded nucleic acid and the helicase using the single molecule fluorescence analysis method. The content of double-stranded nucleic acid ([double-stranded nucleic acid content] / [double-stranded nucleic acid content] + [single-stranded nucleic acid content]) and the apparent double-stranded nucleic acid content obtained by measurement from the measurement sample, By creating an approximate line of the above relationship and using this approximate line, the unwinding reaction efficiency of the double-stranded nucleic acid can be measured more quantitatively and accurately, and the helicase activity can be evaluated more accurately. The present invention has been completed.

That is, the present invention
(1) A method for measuring the unwinding reaction efficiency of a helicase to a double-stranded nucleic acid, wherein (a) a first fluorescently labeled single-stranded nucleic acid labeled with a first fluorescent substance and the first fluorescent substance are After adding helicase to the reaction solution containing the fluorescently labeled double-stranded nucleic acid that is an association with the second fluorescently labeled single-stranded nucleic acid labeled with the second fluorescent substance having different spectral characteristics, The reaction solution is irradiated with light having a wavelength that can excite the first fluorescent substance and light having a wavelength that can excite the second fluorescent substance, and fluorescence emitted from each fluorescent substance is detected as a fluorescent signal, respectively. Analyzing the detected fluorescence signal by single molecule fluorescence analysis and measuring the apparent content of the fluorescently labeled double-stranded nucleic acid in the reaction solution; and (b) labeled with the first fluorescent substance. 1st fluorescent indicator for calibration curve Containing an equal amount of a single-stranded nucleic acid and a second fluorescent-labeled single-stranded nucleic acid for a calibration curve labeled with the second fluorescent substance, and labeled with the first fluorescent substance and the second fluorescent substance The fluorescence-labeled double-stranded nucleic acid for the calibration curve with respect to the sum of the fluorescence-labeled double-stranded nucleic acid for the calibration curve, the first fluorescence-labeled single-stranded nucleic acid for the calibration curve, and the second fluorescence-labeled single-stranded nucleic acid for the calibration curve And (c) the first fluorescent substance can be excited to each of the calibration curve samples of the calibration curve sample series prepared in the step (b). Irradiate light of a wavelength and light of a wavelength that can excite the second fluorescent substance, respectively, detect fluorescence emitted from each fluorescent substance as a fluorescent signal, and analyze the detected fluorescent signal by single molecule fluorescence analysis The calibration curve in the sample for the calibration curve A step of measuring an apparent content ratio (molar ratio) of the fluorescently labeled double-stranded nucleic acid, and (d) an apparent appearance of the fluorescently labeled double-stranded nucleic acid for the calibration curve of each calibration curve sample measured in the step (c). And (e) the approximation created in step (d) above, a step of creating an approximate line of the relationship between the content ratio (molar ratio) of γ and the content of the actual calibration curve fluorescently labeled double-stranded nucleic acid in the measurement sample And calculating the unwinding reaction efficiency of the helicase based on the apparent content ratio (molar ratio) of the fluorescently labeled double-stranded nucleic acid in the reaction solution measured in the step (a). A method for measuring the unwinding reaction efficiency of a helicase, characterized by:
(2) The method for measuring the unwinding reaction efficiency of a helicase according to (1), wherein the single molecule fluorescence analysis method is fluorescence cross-correlation spectroscopy,
(3) The fluorescent substance is a fluorescent substance that is excited by excitation light having a wavelength of 488 nm, and the approximate line created in the step (d) is a fluorescent labeled double-stranded nucleic acid for an actual calibration curve in a measurement sample. It consists of an approximate line in the low region of the content ratio and an approximate line in the high region of the content ratio, and the boundary between the low region and the high region is in the range of 60 to 80%. The method for measuring the unwinding reaction efficiency of the helicase according to (1) or (2),
(4) The method for measuring the unwinding reaction efficiency of the helicase according to (3), wherein a boundary between the low region and the high region is in a range of 60 to 70%,
Is to provide.

  In the method for measuring the unwinding reaction efficiency of a helicase of the present invention, the fluorescently labeled double stranded nucleic acid and the fluorescently labeled single stranded nucleic acid are contained in a reaction solution obtained by mixing and reacting the fluorescently labeled double stranded nucleic acid and helicase. Since the ratio (molar ratio), that is, the residual ratio of the fluorescently labeled double-stranded nucleic acid can be determined with high accuracy, the unwinding reaction efficiency of the helicase can be determined more quantitatively and accurately. Therefore, by using the method for measuring the unwinding reaction efficiency of the helicase of the present invention, more detailed helicase enzyme activities such as substrate specificity and reaction efficiency can be evaluated with high accuracy. In addition, the method for measuring the unwinding reaction efficiency of the helicase of the present invention is a simple and safe method because it uses a single molecule fluorescence analysis method. Further, the detailed efficiency measurement of helicase activity can be performed quickly and in a small amount of sample. Can be done against.

It is the schematic which showed the change of the fluorescence cross correlation function curve and the fluorescence autocross correlation function curve by the unwinding reaction by helicase. A fluorescently labeled double-stranded nucleic acid is labeled with a single-stranded nucleic acid whose 5 ′ end is labeled with a first fluorescent substance (star shape in the figure) and a 5 ′ end with a second fluorescent substance (cross shape in the figure). It is the conceptual diagram which showed the one aspect | mode of the sample series for a calibration curve in the case of being an aggregate with the single stranded nucleic acid. It is the flowchart figure which showed the one aspect | mode of the rewinding reaction efficiency measuring method of this invention at the time of using FCCS as a single molecule fluorescence analysis method. It is the flowchart figure which showed the one aspect | mode of the rewinding reaction efficiency measuring method of this invention at the time of using FIDA as a single molecule fluorescence analysis method. In Example 1, the vertical axis indicates the apparent content (DCC) of fluorescently labeled double-stranded nucleic acid obtained by FCCS measurement, and the horizontal axis indicates the actual content of fluorescently labeled double-stranded nucleic acid (Frac. G / R- (DNA) is a diagram plotting data of each calibration curve sample of the calibration curve sample series. In Example 1, the apparent content ratio (DCC) of the fluorescently labeled double-stranded nucleic acid in the reaction solution after the reaction with helicase is shown for each concentration of the DNA helicase added to the reaction solution, as measured by FCCS measurement. It is a graph. In Example 1, “♦” indicates two fluorescent labels in the reaction solution calculated from the apparent content ratio of the fluorescently labeled double-stranded nucleic acid by the FCCS measurement shown in FIG. 6 based on the calibration curve shown in FIG. It is the graph which showed the content rate (remaining rate of a fluorescence labeled double stranded nucleic acid) (Frac.G / R-DNA) for every density | concentration of the DNA helicase added to the reaction solution. On the other hand, “Δ” is a graph showing the result of measuring the content ratio of the double-stranded nucleic acid in the reaction solution after carrying out the helicase reaction under the same reaction conditions by electrophoresis. In Reference Example 1, the vertical axis represents the content of fluorescently labeled double-stranded nucleic acid (DCC) obtained by FCCS measurement, and the horizontal axis represents the actual content of fluorescently labeled double-stranded nucleic acid (Frac.G / R-DNA). FIG. 8 is a diagram plotting data of each calibration curve sample of the calibration curve sample series. The figure which showed the content rate (Q = 12.7) of the single-sided fluorescent labeling double-stranded nucleic acid in the reaction solution of each helicase concentration in Reference Example 2, and the content rate (Q = 19) of the single-sided fluorescent labeling single-stranded nucleic acid. It is. It is the figure which showed the result of having measured the unwinding reaction efficiency (%) in the reaction solution after performing helicase reaction on the reaction conditions similar to the reference example 2 by the electrophoresis method.

  In the present invention and the specification of the present application, the helicase means all proteins having a property of rewinding a double-stranded nucleic acid into a single-stranded state regardless of the name of the protein. Typically, helicases act using the hydrolysis energy of ATP and GTP. The helicase used in the method for measuring the unwinding reaction efficiency of the helicase of the present invention (hereinafter sometimes referred to as “test helicase”) may be a DNA helicase or an RNA helicase.

  In the present invention and the present specification, unless otherwise specified, “amount” means “molar amount”, “quantity ratio” or “ratio” means “molar ratio”, and “ratio” means “molar ratio”. , “Ratio” means “ratio between molar amounts”, respectively.

  Further, in the present invention and the specification of the present application, the “content ratio (molar ratio) of fluorescently labeled double-stranded nucleic acid” of a solution means the total amount (molar amount) of fluorescently labeled double-stranded nucleic acid in the solution and the fluorescent label. It means the ratio of the total amount (molar amount) of fluorescently labeled double-stranded nucleic acid to the sum of the total amount (molar amount) of single-stranded nucleic acid.

  In the present invention, a fluorescently labeled double-stranded nucleic acid composed of an association of a first fluorescently labeled single-stranded nucleic acid and a second fluorescently labeled single-stranded nucleic acid is used as a substrate for the test helicase. The first fluorescently labeled single-stranded nucleic acid is a single-stranded nucleic acid labeled with a fluorescent substance (first fluorescent substance), and the second fluorescently-labeled single-stranded nucleic acid has spectral characteristics different from that of the first fluorescent substance. It is a single-stranded nucleic acid labeled with a different fluorescent substance (second fluorescent substance). In the present invention and the specification of the present application, “fluorescent substances having different spectral characteristics” mean fluorescent substances that emit fluorescence having different wavelengths.

  The base type, base sequence, base pair length, and the like constituting the fluorescently labeled double-stranded nucleic acid can be appropriately determined in consideration of the type of test helicase. For example, when the test helicase is a DNA helicase, the fluorescently labeled double-stranded nucleic acid is preferably double-stranded DNA. When the test helicase is an RNA helicase, the fluorescently labeled double-stranded nucleic acid is Preferred is a double-stranded RNA or a chimeric double-stranded nucleic acid comprising a DNA strand and an RNA strand. In addition, the fluorescently labeled double-stranded nucleic acid may be modified by phosphorylation or 2'-O-methylation. Moreover, the nucleotide analog generally used may be sufficient as the one part base which comprises a fluorescence labeled double strand nucleic acid.

  In addition, the fluorescently labeled double-stranded nucleic acid may have a blunt end at both ends or a protruding end. For example, it may be a double-stranded nucleic acid whose one end is a 5 'protruding end or 3' protruding end and the other is a smooth end. It can be appropriately determined in consideration of the substrate specificity of the test helicase. In addition, by devising the complementarity and length of the two single-stranded nucleic acids constituting the fluorescence-labeled double-stranded nucleic acid, both-end smooth double-stranded nucleic acid and 5 ′ protruding end double-stranded nucleic acid, 3 ′ protruding end Double stranded nucleic acids can be made.

  The first fluorescent substance or the second fluorescent substance used for labeling a fluorescently labeled double-stranded nucleic acid or the like is not particularly limited as long as the spectral characteristics are different from each other. It can be suitably determined from among the fluorescent materials used in the above. Examples of such fluorescent substances include FAM, TAMRA, Rhodamine Green, Alexa Fluor 488, Alexa Fluor 633, Alexa Fluor 647, etc. Alexa Fluor (registered trademark) dye series (manufactured by Invitrogen), ATTO dy, etc. Series (made by ATTO-TEC), Cydy series such as Cy5 (made by GE Healthcare Biosciences), HiLyte (registered trademark) Fluor488 (made by AnaSpec), BODIPY FL (made by Invitrogen), DY647 (made by Dynamics) ), FITC (fluorescein isothiocyanate), fluorescein, rhodamine, NBD, TMR (tetramethylrhodamine) There is TexasRed like. For example, by using, as a label, a fluorescent material that is relatively stable and emits fluorescence even after continuous light irradiation, such as rhodamine green, TAMRA, TMR, etc. It is possible to suppress the influence of the number of times, prevent variation for each measurement value, and obtain a more stable analysis result.

The first fluorescent material and the second fluorescent material are each preferably a combination of fluorescent materials that do not suffer from the wavelength of fluorescence emitted after excitation. For example, it is preferable to use a green fluorescent material as the first fluorescent material and a red fluorescent material as the second fluorescent material. Examples of the green fluorescent material include fluorescent materials whose excitation light is around 488 nm, specifically, rhodamine green, HiLyte Fluor 488, FITC, Alexa fluor 488, FAM, and Bodipy FL. On the other hand, examples of the red fluorescent material include fluorescent materials having excitation light of 590 nm or more, specifically Texas Red, ATTO 633, Alexa fluor 647, Alexa fluor 633, Cy5, and the like.
The first fluorescent material and the second fluorescent material are preferably a combination that does not cause FRET between the two fluorescent materials. This is because when FRET occurs between two types of fluorescent substances that label fluorescently labeled double-stranded nucleic acids, the accuracy of FCCS analysis is reduced.

  Further, the base pair length of the fluorescently labeled double-stranded nucleic acid is not particularly limited as long as it is long enough to serve as a substrate for the unwinding reaction by the test helicase. In the present invention, it is possible to improve the discrimination accuracy between the double-stranded nucleic acid before being unwound and the single-stranded nucleic acid after being unwound. More preferably. In particular, when an aggregate of single-stranded nucleic acids labeled with fluorescent substances having different spectral characteristics is used as the fluorescent-labeled double-stranded nucleic acid, FRET is prevented from occurring between the fluorescent substances used for labeling. Therefore, the length of the fluorescently labeled double-stranded nucleic acid is preferably 20 base pairs or more.

  If the sites in the fluorescently labeled double-stranded nucleic acid, the first fluorescently labeled single-stranded nucleic acid, and the second fluorescently labeled single-stranded nucleic acid that are labeled with a fluorescent substance are regions that do not inhibit unwinding by the test helicase, It is not particularly limited, and can be appropriately determined in consideration of the fluorescent labeling method, the type of single-molecule fluorescence analysis method used, and the like. In the present invention, it is preferable that a fluorescent substance is labeled at the 5 ′ end of the single-stranded nucleic acid, but when producing a 5 ′ protruding end double-stranded nucleic acid or a 3 ′ protruding end double-stranded nucleic acid, You may devise the place to label suitably.

  The fluorescently labeled double-stranded nucleic acid, the first fluorescently labeled single-stranded nucleic acid, and the second fluorescently labeled single-stranded nucleic acid used in the present invention may be designed and synthesized by any method known in the art. . For example, each fluorescently labeled single-stranded nucleic acid can be synthesized by a known nucleic acid synthesis reaction using a commercially available synthesizer or the like after designing the base sequence by a conventional method. In addition, it is also possible to synthesize using a contract service. On the other hand, fluorescently labeled double-stranded nucleic acids can be synthesized, for example, by annealing single-stranded nucleic acids synthesized separately. After annealing, it is preferable to remove the remaining single-stranded nucleic acid by exonuclease treatment or the like.

  In addition, a double-stranded nucleic acid can be synthesized using a nucleic acid amplification reaction such as PCR (Polymerase Chain Reaction). The synthesized double-stranded nucleic acid may be used after separation and purification based on molecular weight using electrophoresis or the like. A single-stranded nucleic acid can also be obtained by denaturing the double-stranded nucleic acid synthesized in this way.

  In addition, for example, a single-stranded nucleic acid is synthesized by using a primer having a fluorescent substance bonded to the 5 ′ end, thereby fluorescently labeled single-stranded nucleic acid or fluorescently labeled double-stranded nucleic acid in which the 5 ′ end is fluorescently labeled. Can be obtained. In particular, by performing PCR (Polymerase Chain Reaction) using two types of primers in which different types of fluorescent substances are bonded to the 5 ′ end, single-stranded nucleic acids labeled with fluorescent substances having different spectral characteristics can be obtained. A fluorescently labeled double-stranded nucleic acid that is an aggregate can be easily prepared.

The method for measuring the unwinding reaction efficiency of the helicase of the present invention (hereinafter sometimes referred to as the method of measuring the unwinding reaction efficiency of the present invention) has the following steps.
(A) a first fluorescently labeled single-stranded nucleic acid labeled with a first fluorescent substance, and a second fluorescently labeled single-stranded nucleic acid labeled with a second fluorescent substance having a spectral characteristic different from that of the first fluorescent substance; Light having a wavelength capable of exciting the first fluorescent substance and the second fluorescent substance in the reaction solution after adding helicase to the reaction solution containing the fluorescently labeled double-stranded nucleic acid that is an aggregate of The fluorescence emitted from each fluorescent substance is detected as a fluorescence signal, and the detected fluorescence signal is analyzed by a single molecule fluorescence analysis method to detect the fluorescence in the reaction solution. Measuring the apparent content of labeled double-stranded nucleic acid;
(B) An equal amount of a first fluorescent-labeled single-stranded nucleic acid for a calibration curve labeled with the first fluorescent substance and a second fluorescent-labeled single-stranded nucleic acid for a calibration curve labeled with the second fluorescent substance And a calibration curve fluorescent labeled double-stranded nucleic acid labeled with the first fluorescent substance and the second fluorescent substance, the calibration curve first fluorescent labeled single-stranded nucleic acid, and the calibration curve second fluorescent label. Preparing a calibration curve sample series in which the content ratio of the fluorescence-labeled double-stranded nucleic acid for calibration curve is different from the total with the single-stranded nucleic acid;
(C) Light of a wavelength that can excite the first fluorescent substance and light of a wavelength that can excite the second fluorescent substance to each of the calibration curve samples of the calibration curve sample series prepared in the step (b). , Respectively, and the fluorescence emitted from each fluorescent substance is detected as a fluorescence signal, the detected fluorescence signal is analyzed by a single molecule fluorescence analysis method, and the calibration curve fluorescent label 2 in the calibration curve sample is analyzed. A step of measuring an apparent content ratio (molar ratio) of the double-stranded nucleic acid;
(D) The apparent content ratio of the fluorescent-labeled double-stranded nucleic acid for the calibration curve of each calibration-line sample measured in the step (c), and the actual content of the fluorescent-labeled double-stranded nucleic acid for the calibration curve in the measurement sample Creating an approximate line of the relationship with the ratio;
(E) Based on the approximate line created in the step (d) and the apparent content ratio of the fluorescently labeled double-stranded nucleic acid in the reaction solution measured in the step (a), the unwinding reaction efficiency of the helicase is determined. The process of calculating.

  In the present invention, the results of measurement / analysis of the content ratio of the actual fluorescent-labeled double-stranded nucleic acid for calibration curve in the measurement sample and the fluorescence signal detected from the measurement sample by steps (b) to (d) An approximate line of the relationship with the apparent content ratio (measured value) of the fluorescently labeled double-stranded nucleic acid for the calibration curve obtained in step 1 is prepared, and the reaction by the test helicase obtained in step (a) based on the approximate line From the apparent content ratio (measured value) of the fluorescently labeled double-stranded nucleic acid in the subsequent reaction solution, the actual fluorescently labeled double-stranded nucleic acid content ratio is calculated. In the present invention, by using such an approximate line, the content ratio of the fluorescently labeled double-stranded nucleic acid in the measurement sample (that is, the residual ratio of the fluorescently labeled double-stranded nucleic acid) and the content calculated from this content ratio. The unwinding reaction efficiency by the helicopter detection can be determined more quantitatively and accurately. In particular, when the fluorescence signal is analyzed by FCS as described in Patent Document 3, when the content ratio of the double-stranded nucleic acid in the reaction solution is low (for example, 30% or less), 2 Although the measurement accuracy of the content ratio of the single-stranded nucleic acid is lowered, in the present invention, the measurement value obtained by measuring and analyzing the fluorescence signal is corrected using the approximate line created in advance, so that it is contained. Even when the amount of double-stranded nucleic acid is small, the content ratio of double-stranded nucleic acid can be determined with high accuracy in the same manner as when the amount of double-stranded nucleic acid contained is large. Regardless of strength, the unwinding reaction efficiency of helicase can be accurately measured.

Hereinafter, it demonstrates for every process.
First, as step (a), a test helicase is added to and reacted with a reaction solution containing a fluorescently labeled double-stranded nucleic acid, and then light having a wavelength capable of exciting the first fluorescent substance and 2 irradiate light of a wavelength that can excite the fluorescent substance, detect fluorescence emitted from each fluorescent substance as a fluorescent signal, analyze the detected fluorescent signal by single molecule fluorescence analysis, The apparent content of the fluorescently labeled double-stranded nucleic acid is measured.

  The reaction solution used in the step (a) is a solution containing a fluorescently labeled double-stranded nucleic acid, to which reagents necessary for a rewinding reaction by a test helicase such as ATP are added, and the test helicase is an enzyme. The reaction solution is not particularly limited as long as it can function as an appropriate one, and can be appropriately determined in consideration of the type of test helicase.

In the present invention, the reaction solution used in step (a) is preferably a solution in which a reagent necessary for helicase activity such as ATP is added to a buffer. This is because depending on the type of fluorescent substance used for labeling the fluorescently labeled double-stranded nucleic acid, it may be easily affected by pH or 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 a nucleolytic enzyme inhibitor or the like is added to the reaction solution used in the step (a).
Further, the amount of the reaction solution and the concentration of the fluorescently labeled double-stranded nucleic acid in the reaction solution are not particularly limited, and should be appropriately determined in consideration of the apparatus used for measurement, the type of fluorescent substance, and the like. Can do.

  The reaction time for reacting the fluorescently labeled double-stranded nucleic acid with the test helicase is not particularly limited as long as it is sufficient for the fluorescently labeled double-stranded nucleic acid to be unwound by the test helicase. It can be determined appropriately in consideration of the type of helicase, the type of reaction solution, the reaction temperature, the concentration of the fluorescently labeled double-stranded nucleic acid, and the like. For example, at room temperature to 37 ° C. (for example, 15 to 37 ° C.), it is preferably about 5 minutes to 2 hours, more preferably about 5 minutes to 1 hour, and further preferably about 10 to 30 minutes. In order to control the reaction time, it is also preferable to stop the helicase reaction by adding a chelating agent such as EDTA to the reaction solution during the predetermined reaction time.

  Next, the reaction solution after the reaction is irradiated with excitation light of the fluorescent material used for labeling, and a fluorescent signal is detected. Specifically, the fluorescence signal can be detected by installing the reaction solution in a fluorometer having a confocal optical system and detecting the fluorescence signal by a conventional method. In addition, the detection of the fluorescence signal analyzed by the single molecule fluorescence analysis method generally uses a confocal optical system, but the fluorescence signal derived from one molecule for performing the single molecule fluorescence analysis is acquired. The optical system is not particularly limited as long as it can be used, and other optical systems other than the confocal optical system may 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 for 10 to 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 short, such as less than 10 seconds, or if the number of measurements is only about once, the reproducibility and reliability of data may be reduced. There is. In addition, by scanning the focal position of a sample for measuring a 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 cross-correlation spectroscopy (FCCS).
FCCS performs cross-correlation function analysis of fluorescently labeled molecules with different spectroscopic characteristics, so that each fluorescently labeled molecule moves synchronously (movement with cross-correlation) or dissociates and moves randomly (movement without cross-correlation). ) Is a technique that can be distinguished. By irradiating the measurement sample with excitation light corresponding to each fluorescent substance at the same time and analyzing the cross-correlation of the fluorescence signals detected from each molecule, it is confirmed whether the movement of different molecules is synchronized. be able to. Since this analysis method looks at the degree of synchronization of the fluorescence signal from each fluorescent dye, it is basically unaffected by the size of each fluorescently labeled molecule. For this reason, when the change in the molecular size before and after the interaction is small, for example, the base pair length of the fluorescently labeled double-stranded nucleic acid is relatively short, and the molecular size between the double-stranded nucleic acid and the unwound single-stranded nucleic acid Even if the change in is small, it is possible to analyze the intermolecular interaction suitably without requiring an enzyme treatment or the like that divides the single-stranded nucleic acid by exonuclease to reduce the molecular weight.

  Specifically, after reacting the fluorescence-labeled double-stranded nucleic acid with the test helicase, each fluorescent substance is irradiated with light having a wavelength that can be excited to detect a fluorescent signal. Fluorescent signals are separated into fluorescent signals emitted from each fluorescent substance using DM (dichroic mirror), and time-series changes in the fluorescence intensity are detected with a highly sensitive detector, and the cross-correlation of each fluorescent signal. Ask for. In the fluorescence-labeled double-stranded nucleic acid, the fluorescent substances labeled with the single-stranded nucleic acids are moving at the same time, so that the cross-correlation between the fluorescent signal from the first fluorescent substance and the fluorescent signal from the second fluorescent substance is strong. On the other hand, when the fluorescently labeled double-stranded nucleic acid is unwound by the test helicase, it becomes two independent single-stranded nucleic acids (first fluorescently-labeled single-stranded nucleic acid and second fluorescently-labeled single-stranded nucleic acid) and reacts. Since the solution moves apart in the solution, the cross-correlation between the fluorescent signals of the fluorescent substances is weakened. For this reason, the binding ratio of each fluorescently labeled molecule (the ratio of molecules moving in synchronization) is calculated from the results of autocorrelation function analysis and cross-correlation function analysis of the fluorescence signal of each fluorescent substance. Can measure the content ratio of the fluorescently labeled double-stranded nucleic acid in the reaction solution.

  FIG. 1 shows a first fluorescently labeled single-stranded nucleic acid using a green fluorescent substance as a first fluorescent substance as a substrate for a test helicase, and a second fluorescently labeled single strand using a red fluorescent substance as a second fluorescent substance. It is the schematic which showed the change of the fluorescence cross-correlation function curve and the fluorescence auto-correlation function curve by the unwinding reaction by helicase at the time of using the fluorescence labeled double stranded nucleic acid which is an aggregate | assembly which consists of nucleic acids. FIG. 1 (A) shows the result of FCCS measurement for a solution containing no fluorescently labeled double-stranded nucleic acid without adding helicase, and FIG. 1 (B) shows the reaction in which helicase was added and reacted. The result of the FCCS measurement with respect to a solution is shown. FIG. 1 (C) is a diagram schematically showing a fluorescence-labeled double-stranded nucleic acid in the absence of helicase, and FIG. 1 (D) is a schematic diagram of the fluorescence-labeled double-stranded nucleic acid after reaction with helicase. It is the figure shown in. In FIGS. 1C and 1D, the star shape indicates a green fluorescent material, the cross shape indicates a red fluorescent material, the oval shape indicates a helicase, and the straight line indicates a nucleic acid chain.

  In a solution without helicase, as shown in FIG. 1C, only the fluorescently labeled double-stranded nucleic acid is present, so that the green fluorescent substance and the red fluorescent substance are always present in the same molecule. Therefore, as shown in FIG. 1A, the autocorrelation curve of the green fluorescent material and the autocorrelation curve of the red fluorescent material substantially coincide, and the Y-intercept of the cross-correlation curves of both fluorescent materials is large. On the other hand, in the reaction solution after the reaction with helicase, as shown in FIG. 1 (D), the single-stranded nucleic acid labeled with the green fluorescent substance and the single-stranded nucleic acid labeled with the red fluorescent substance are separated. Move as another molecule. Therefore, as shown in FIG. 1B, the autocorrelation curve of the green fluorescent material and the autocorrelation curve of the red fluorescent material are dissociated, and the Y-intercept of the cross-correlation curve of both fluorescent materials is shown in FIG. It can be seen that the cross correlation is low.

As a substrate of the test helicase, it consists of a first fluorescently labeled single-stranded nucleic acid using a green fluorescent substance as a first fluorescent substance and a second fluorescently labeled single-stranded nucleic acid using a red fluorescent substance as a second fluorescent substance. Specifically, the content ratio of the fluorescently labeled double-stranded nucleic acid in the reaction solution when the fluorescently labeled double-stranded nucleic acid that is an aggregate is used can be obtained as follows. First, the number of molecules (the number of molecules of fluorescently labeled double-stranded nucleic acid) to which the green fluorescent substance and the red fluorescent substance in the reaction solution are bonded is N _gr , and the number of unlabeled labeled molecules is N _g (covered). Nr (number of molecules of single-stranded nucleic acid labeled with red fluorescent material unwound by test helicase), N _r (number of molecules of single-stranded nucleic acid labeled with green fluorescent material unwound by test helicase) and To do. Among the molecules present in the reaction solution, the molecular content (DCC) of the fluorescently labeled double-stranded nucleic acid is the single-stranded nucleic acid labeled with the green fluorescent substance and the single labeled with the red fluorescent substance. This is the rate at which the chain nucleic acid is bound, and is represented by the following formula (1).
Formula (1) DCC (%) = 100 × N _gr / (N _gr + N _g + N _r )

First, autocorrelation function analysis is performed from a fluorescent signal emitted from a green fluorescent substance, and the number of fluorescent molecules N _Gato is calculated. In addition, autocorrelation function analysis is performed from the fluorescent signal emitted from the red fluorescent substance, and the number of fluorescent molecules N _Rato is calculated. Further, a cross-correlation function analysis between the fluorescence from the green fluorescent material and the fluorescence from the red fluorescent material is performed, and N _cross is calculated. When the parameters produced in this way are used, the relationship represented by the following formula (2) is established.

In the above formula, N _Gauto is the sum of N _gr and N _g [N _gr + N _g ], and N _Rato is the sum of N _gr and N _r [N _gr + N _r ]. Therefore, based on the analysis result, N _gr , N _g , and N _r are calculated from the above formula (“Fluorescence correlation spectroscopy in nucleic acid analysis”, Fluorescence correlation spectroscopy-theory and applications, R. Rigler, ESElson, Springer Series in Chemical physics 65, p33-35).

  Further, in the present invention, the results of measuring and analyzing the content ratio of the actual fluorescently labeled double-stranded nucleic acid in the measurement sample and the fluorescence signal detected from the measurement sample by steps (b) to (d). An approximate line (hereinafter sometimes referred to as “calibration curve”) of the relationship with the apparent content ratio (measured value) of the fluorescently labeled double-stranded nucleic acid obtained is prepared.

  As described above, FCCS has a fundamental problem that the bonding rate of two kinds of molecules cannot be accurately measured due to the problem that the irradiation region of the sample is shifted for each wavelength due to chromatic aberration of the objective lens. I have it. On the other hand, in the present invention, by using the calibration curve prepared in steps (b) to (d), the content ratio of the fluorescently labeled double-stranded nucleic acid in the reaction solution (that is, the fluorescently labeled double-stranded nucleic acid) The remaining rate of the image can be obtained more accurately. Therefore, when FCCS is used as the single molecule fluorescence analysis method, the measurement accuracy of the unwinding reaction efficiency of the helicase can be dramatically improved by using a calibration curve.

  Specifically, first, as step (b), a fluorescently labeled double-stranded nucleic acid (fluorescent labeled double-stranded nucleic acid for calibration curve) and a fluorescently labeled single-stranded nucleic acid (fluorescent label for calibration curve) for preparing a calibration curve Single-stranded nucleic acid) is prepared, and these are mixed as appropriate to prepare a calibration curve sample series in which the content ratio of the fluorescent-labeled double-stranded nucleic acid for calibration curve is different. The number of calibration curve samples constituting the calibration curve sample series is not particularly limited, and is preferably about 3 to 20 types. In addition, the content of the calibration-label fluorescent-labeled double-stranded nucleic acid in each calibration-line sample is as follows: the calibration-line sample in which the content of the calibration-label fluorescent-labeled double-stranded nucleic acid is 0% and the calibration curve sample of 100% As long as it contains, it is not particularly limited, and can be appropriately determined in consideration of the type of fluorescent substance used for labeling, the type of single-molecule fluorescence analysis method, and the like. For example, 11 types of calibration curve sample series in which the content of the fluorescent labeled double-stranded nucleic acid for calibration curve is 0, 10, 20, 30, 40, 50, 60, 70, 80, 90, and 100% In addition, the content of the fluorescent-labeled double-stranded nucleic acid for calibration curve of each calibration curve sample may be set at equal intervals, 0, 10, 20, 40, 50, 80, and 100% As in the seven types of calibration curve sample series, the calibration curve fluorescent-labeled double-stranded nucleic acid content of each calibration curve sample may be set at unequal intervals. “The content of the fluorescently labeled double-stranded nucleic acid for the calibration curve is 0%” means that the calibration label fluorescently labeled double-stranded nucleic acid is not present in the solution and only the fluorescently labeled single-stranded nucleic acid is present. On the contrary, “the content ratio of the fluorescent labeled double-stranded nucleic acid for calibration curve is 100%” means that only the fluorescent labeled double-stranded nucleic acid for calibration curve exists in the solution and there is no fluorescent labeled single-stranded nucleic acid. It is a solution.

  The fluorescently labeled double-stranded nucleic acid for the calibration curve may be a double-stranded nucleic acid in which the same position in the nucleic acid chain is labeled with the fluorescently labeled double-stranded nucleic acid used in step (a) and the same type of fluorescent substance. For example, there is no particular limitation. For example, the fluorescently labeled double-stranded nucleic acid used in step (a) is a single-stranded nucleic acid whose 5 ′ end is labeled with a first fluorescent substance and a single strand whose 5 ′ end is labeled with a second fluorescent substance. In the case of an associated body with a nucleic acid, the fluorescent-labeled double-stranded nucleic acid for calibration curve has two ends labeled with a first fluorescent substance and the other end labeled with a second fluorescent substance. It may be a strand nucleic acid, and its base sequence, base pair length, etc. may be different from the fluorescently labeled double-stranded nucleic acid used in step (a). In the present invention, since a calibration curve with higher accuracy can be created, the fluorescent-labeled double-stranded nucleic acid for the calibration curve may be the same as the fluorescent-labeled double-stranded nucleic acid used in step (a). preferable.

  The single-stranded nucleic acid used in the sample for the calibration curve is labeled at the same position in the nucleic acid chain by the single-stranded nucleic acid constituting the fluorescent-labeled double-stranded nucleic acid used in step (a) and the same type of fluorescent substance. As long as it is a single-stranded nucleic acid, it is not particularly limited, and may have different base sequences, base lengths, and the like. In the present invention, a single-stranded nucleic acid labeled with the first fluorescent substance at the same position as the first fluorescent-labeled single-stranded nucleic acid is referred to as a first fluorescent-labeled single-stranded nucleic acid for a calibration curve, and the second fluorescent substance The single-stranded nucleic acid labeled at the same position as the second fluorescently labeled single-stranded nucleic acid is referred to as a second fluorescent-labeled single-stranded nucleic acid for calibration curve. In the present invention, since a calibration curve with higher accuracy can be prepared, the first fluorescent labeled single-stranded nucleic acid for calibration curve and the second fluorescent labeled single-stranded nucleic acid for calibration curve are the fluorescent label 2 for calibration curve. The single-stranded nucleic acid obtained by cleaving the double-stranded nucleic acid is preferably one that does not form an aggregate (non-annealed) due to having a non-complementary base sequence or the like.

  Each sample for the calibration curve contains equal amounts of the first fluorescence-labeled single-stranded nucleic acid for calibration curve and the second fluorescence-labeled single-stranded nucleic acid for calibration curve. That is, each calibration curve sample has a content ratio of the first fluorescent labeled single-stranded nucleic acid for calibration curve of [(1-X) when the content ratio of the fluorescent labeled double-stranded nucleic acid for calibration curve is X%. / 2]%, and the content ratio of the second fluorescently labeled single-stranded nucleic acid for calibration curve is [(1-X) / 2]%.

  FIG. 2 shows a fluorescence-labeled double-stranded nucleic acid for a calibration curve, a single-stranded nucleic acid labeled at the 5 ′ end with a first fluorescent substance (star shape in the figure) and a second fluorescent substance at the 5 ′ end (in the figure). FIG. 2 is a conceptual diagram showing one embodiment of a sample series for a calibration curve in the case of an aggregate with a single-stranded nucleic acid labeled with a cross shape. 2 (A) to 2 (E) schematically show the content ratios of nucleic acids in the calibration curve samples. 2A is 100%, FIG. 2B is 50%, FIG. 2C is 33.3%, and the content of the fluorescent-labeled double-stranded nucleic acid for the calibration curve is shown in FIG. 2 (D) is 16.7% and FIG. 2 (E) is 0%.

  The composition and pH of each calibration curve sample constituting the calibration curve sample series are adjusted in the same manner as the composition and pH of the reaction solution in step (a). This is because the detected fluorescent signal may be affected by the composition and pH of the measurement sample depending on the type of fluorescent substance.

  Next, as the step (c), the apparent content ratio of the calibration-label fluorescent-labeled double-stranded nucleic acid in each calibration curve sample of the calibration curve sample series prepared in the step (b) is measured. Specifically, in the same manner as in step (a), each calibration curve sample is irradiated with light having a wavelength capable of exciting the first fluorescent substance and light having a wavelength capable of exciting the second fluorescent substance, respectively. Fluorescence emitted from the fluorescent substance is detected as a fluorescence signal, and the detected fluorescence signal is analyzed by a single molecule fluorescence analysis method. The apparatus used for fluorescence signal detection, the volume of the sample solution used for measurement, the measurement temperature, the wavelength and intensity of excitation light, the irradiation conditions, the detection conditions for the fluorescence signal, the analysis method and conditions for the detected signal, etc. Perform under the same conditions as a).

  Thereafter, as step (d), the apparent content ratio of the calibration-label fluorescent-labeled double-stranded nucleic acid in each calibration-sample sample measured in step (c), and the actual calibration-label fluorescence-labeled double-strand in the measurement sample A calibration curve is created from the nucleic acid content. The method for creating the calibration curve is not particularly limited, and it may be created using any of the arithmetic analysis methods used for creating the approximate line of the relationship between the two types of quantitative data. For example, the horizontal axis represents the content (%) of the fluorescence-labeled double-stranded nucleic acid for the calibration curve of each measurement sample, and the vertical axis represents the content (%) of the fluorescence-labeled double-stranded nucleic acid for the calibration curve measured. The data obtained by the step (c) is plotted on the graph. A calibration curve can be created by performing a least square method or the like on each plot.

  The calibration curve is preferably a straight line, but it is not always necessary to approximate the entire measurement range to a single straight line. For example, as shown in Example 1 below, when a double-stranded nucleic acid used as a helicase substrate is labeled with a fluorescent substance having an excitation wavelength of 488 nm, the fluorescent label for the actual calibration curve in the measurement sample is used. It is preferable to create approximate lines composed of straight lines separately for the region with a low content of double-stranded nucleic acid and the region with a high content. In this way, an approximate line composed of two straight lines, that is, an approximate line in the low region of the content ratio of the fluorescent labeled double-stranded nucleic acid for the actual calibration curve in the measurement sample and an approximate line in the high region of the content ratio is calibrated. Even when a fluorescent substance having different fluorescence intensity emitted from a fluorescent substance per molecule is used in the state of a single-stranded nucleic acid and the state of a double-stranded nucleic acid by using a line, in step (a) From the obtained measurement value, the content ratio of the actual fluorescent-labeled double-stranded nucleic acid for calibration curve can be determined with higher accuracy.

  When creating an approximate straight line by dividing the content of the fluorescently labeled double-stranded nucleic acid in the measurement sample into the low and high regions, the boundary between the low and high regions depends on the type of fluorescent substance. It can be determined as appropriate. For example, when a fluorescent substance having an excitation wavelength of 488 nm is used, the boundary between the low region and the high region is such that the content ratio of the fluorescent-labeled double-stranded nucleic acid for the calibration curve is in the range of 60 to 80%. Is preferably set to be in the range of 60 to 70%. The “boundary between the low region and the high region” specifically refers to an approximate straight line created from a plot in the low region of the content ratio of the fluorescent labeled double-stranded nucleic acid for the calibration curve and a plot in the high region. Indicates the point where the approximate line created from

  In addition, process (a) and process (b)-(d) should just be performed before the process (e), respectively, and any may be performed first. For example, the steps (b) to (d) may be sequentially performed after the step (a) is performed, or the step (a) may be performed after the steps (b) to (d) are performed. Furthermore, the operation from the irradiation of the excitation light to the reaction solution in the step (a) to the detection of the fluorescence signal is the operation from the irradiation of the excitation light to each calibration curve sample to the detection of the fluorescence signal in the step (c). You may go at the same time.

  The calibration curve mainly depends on the type and combination of the first fluorescent substance and the second fluorescent substance, the position in the nucleic acid chain labeled with the fluorescent substance, and the like. In addition, when the measurement conditions for single molecule fluorescence analysis are the same, the variation in the measured value for each experiment is small. For this reason, it is not always necessary to perform steps (b) to (d) for each type of fluorescently labeled double-stranded nucleic acid, and another fluorescent label 2 in which the same position in the nucleic acid chain is labeled with the same type of fluorescent substance. A calibration curve already created for the main chain nucleus can also be used.

  Thereafter, as step (e), the unwinding reaction efficiency of the test helicase is calculated. The content ratio of the fluorescently labeled double-stranded nucleic acid in the reaction solution after the reaction with helicase is the ratio of the fluorescently labeled double-stranded nucleic acid that has not undergone the unwinding reaction with helicase (the remaining ratio of the fluorescently labeled double-stranded nucleic acid). . That is, the amount of fluorescently labeled double-stranded nucleic acid that has decreased after the reaction becomes the amount of double-stranded nucleic acid that has undergone the unwinding reaction by helicase.

  Specifically, based on the calibration curve created in step (d), the value of the apparent content ratio of the fluorescently labeled double-stranded nucleic acid in the reaction solution measured in step (a) is corrected, and the reaction solution contains The actual content ratio of the fluorescence-labeled double-stranded nucleic acid (remaining ratio of the fluorescence-labeled double-stranded nucleic acid) is calculated. By subtracting the residual ratio (%) of the obtained fluorescently labeled double-stranded nucleic acid from 100%, the unwinding reaction efficiency of the test helicase can be calculated.

  FIG. 3 is a flowchart showing one embodiment of the method for measuring the rewinding reaction efficiency of the present invention when FCCS is used as the single molecule fluorescence analysis method. In addition, it cannot be overemphasized that the rewinding reaction efficiency measuring method of this invention is not limited to these aspects.

  The flowchart of FIG. 3 will be described. In this embodiment, the same fluorescently labeled double-stranded nucleic acid as the substrate for the test helicase is used as the fluorescently labeled double-stranded nucleic acid for the calibration curve used for preparing the calibration curve, and the steps (b) to (b) After preparing the calibration curve by (d), the reaction is carried out using the test helicase as step (a). First, fluorescence that is an association of a single-stranded nucleic acid labeled with a first fluorescent substance at the 5 ′ end and a single-stranded nucleic acid labeled with a second fluorescent substance at the 5 ′ end as a substrate for the test helicase. A labeled double-stranded nucleic acid (in the figure, “both-end fluorescently labeled double-stranded nucleic acid”) is prepared (step 01). Next, a single-stranded nucleic acid labeled with a first fluorescent substance at the 5 ′ end (first fluorescent labeled single-stranded nucleic acid for detection) and a single-stranded nucleic acid labeled with a second fluorescent substance at the 5 ′ end (detection) (Second fluorescently labeled single-stranded nucleic acid for use) ("One-side fluorescently labeled single-stranded nucleic acid (2 types)" in the figure) (step 02). The two types of single-sided fluorescently labeled single-stranded nucleic acids have the same base length as the single-stranded nucleic acid obtained by cleaving the double-sided fluorescently labeled double-stranded nucleic acid, and have a base sequence that does not anneal with them. It is preferable to design and synthesize. Next, these three types of nucleic acids are mixed as appropriate to prepare a calibration curve sample series [step (b)] (step 03). FCCS measurement is performed on each of these calibration curve samples, and the apparent content ratio of the fluorescently labeled double-stranded nucleic acid (“binding ratio DCC” in the figure) is measured [step (c)] (step 04), A calibration curve is created based on the measurement result [step (d)] (step 05). Thereafter, both-end fluorescently labeled double-stranded nucleic acid and test helicase are mixed and reacted (step 06), FCCS measurement is performed on the reaction solution after the reaction, and the apparent content ratio of fluorescently labeled double-stranded nucleic acid ( In the figure, “binding ratio DCC”) is measured [step (a)] (step 07). Referring to the measurement results and the prepared calibration curve (step 08), the actual content ratio of fluorescently labeled double-stranded nucleic acid (fluorescent label 2) from the apparent content ratio of fluorescently labeled double-stranded nucleic acid in the reaction solution. (Remaining rate of single-stranded nucleic acid) is calculated (step 09), and the unwinding reaction efficiency of the test helicase is calculated [step (e)] (step 10).

  The unwinding reaction efficiency measurement method of the present invention can measure the unwinding reaction efficiency of helicase very quantitatively and accurately, so that the substrate specificity and the reaction efficiency of multiple types of helicases can be compared in more detail. In addition, the enzymatic activity of helicase can be evaluated. For example, using a fluorescently labeled double-stranded nucleic acid having a different terminal shape, base sequence type, length, etc. as a substrate, measuring the unwinding reaction efficiency of the test helicase by the unwinding reaction efficiency measuring method of the present invention, By comparing these, a highly reliable result can be obtained for the substrate specificity of the test helicase. In addition, the unwinding reaction efficiency measurement method of the present invention was performed on the same reaction conditions for multiple types of helicases, and by comparing the obtained unwinding reaction efficiencies, the presence or absence of collarcase activity and the strength of helicase activity were determined. It can also be judged.

  Also, when a fluorescence intensity distribution analysis method (FIDA) or a fluorescence polarization analysis method (FIDA-PO) is used as a single molecule fluorescence analysis method, a calibration curve is prepared in advance, and a test helicase is based on the calibration curve. By calculating the actual content of fluorescently labeled double-stranded nucleic acid from the apparent content (measured value) of fluorescently labeled double-stranded nucleic acid in the reaction solution after the reaction according to, two fluorescent labels in the reaction solution The content ratio of the strand nucleic acid can be determined more accurately.

  FIDA is a technique that can determine the fluorescence intensity per molecule and the number thereof in a reaction solution. If there are fluorescently labeled molecules with different fluorescence intensities in the observation region (ie, molecules with different sizes), the number and ratio of molecules of each size are calculated by performing a two-component analysis. It is also possible to do. For example, fluorescently labeled oligonucleotides have different fluorescence intensity per molecule depending on the base sequence, base length, environmental state of fluorescent dyes such as single-stranded nucleic acid and double-stranded nucleic acid. For this reason, there is a possibility that the fluorescence intensity of the fluorescently labeled single-stranded nucleic acid obtained by unwinding the fluorescently labeled double-stranded nucleic acid changes as compared with the fluorescently labeled double-stranded nucleic acid. Therefore, by analyzing using FIDA, from the obtained fluorescence intensity, whether the fluorescently labeled molecule present in the reaction solution is a double-stranded nucleic acid is unwound by the test helicase and cleaved 1 Whether the nucleic acid is a double-stranded nucleic acid can be distinguished and identified.

  On the other hand, 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 the fluorescence-labeled molecule can be obtained. The degree of fluorescence polarization varies depending on the size of the molecule. Therefore, by measuring the degree of fluorescence polarization of each molecule, whether the fluorescently labeled molecule present in the reaction solution is a double-stranded nucleic acid or not by a test helicase. It is possible to distinguish and identify whether it is a single-stranded nucleic acid that has been unwound.

  Thus, even when analyzed by FIDA or FIDA-PO, the number and abundance of double-stranded nucleic acids and single-stranded nucleic acids in the reaction solution can be calculated. The content ratio of the labeled double-stranded nucleic acid can be measured. In addition, the fluorescence intensity and fluorescence polarization degree of the fluorescence signal detected from the reaction solution are compared with the fluorescence intensity and fluorescence polarization degree of each of the fluorescence-labeled double-stranded nucleic acid and the fluorescence-labeled single-stranded nucleic acid measured in advance. Thus, it can be determined whether the molecule in the reaction solution is a double-stranded nucleic acid or a single-stranded nucleic acid. In particular, in the case of FIDA measurement, since the measurement time per sample can be performed in about 1 second and the reproducibility is good, analysis using FIDA is particularly suitable for screening and the like.

  The calibration curve sample series is composed of a plurality of calibration curve samples in which the content ratio of the fluorescently labeled double-stranded nucleic acid to the sum of the fluorescently labeled double-stranded nucleic acid and the fluorescently labeled single-stranded nucleic acid is different. . Here, in the case of FIDA or the like, unlike the FCCS, only fluorescence emitted from one type of fluorescent material is analyzed. For this reason, the calibration curve sample series includes a double-stranded nucleic acid labeled with both the first fluorescent substance and the second fluorescent substance, and a single-stranded nucleic acid labeled only with the first fluorescent substance, as in the case of FCCS. And a single-stranded nucleic acid labeled only with the second fluorescent substance, or a nucleic acid labeled only with the fluorescent substance to be analyzed may be used. For example, when analyzing a fluorescent signal detected from a first fluorescent substance, the sum of a double-stranded nucleic acid labeled only with the first fluorescent substance and a single-stranded nucleic acid labeled only with the first fluorescent substance It may be a sample series for a calibration curve in which the content ratio of the double-stranded nucleic acid is different.

  In addition, how the fluorescence intensity per molecule and the degree of fluorescence polarization change between single-stranded nucleic acid and double-stranded nucleic acid varies greatly depending on the type of fluorescent substance. This is because the content ratio of the fluorescently labeled double-stranded nucleic acid in the reaction solution can be determined more accurately by creating a calibration curve.

  FIG. 4 is a flowchart showing one mode when FIDA is used as a single molecule fluorescence analysis method. In this embodiment, after preparing a calibration curve in advance, the reaction is performed using the test helicase as step (a). First, a calibration curve is created (step 100). Next, a fluorescently labeled double-stranded nucleic acid that is an association of a single-stranded nucleic acid whose 5 ′ end is labeled with a first fluorescent substance and an unlabeled single-stranded nucleic acid as a substrate for the test helicase (in the figure, “One-sided fluorescently labeled double-stranded nucleic acid”) is prepared (step 101). Further, a single-stranded nucleic acid whose 5 ′ end is labeled with a first fluorescent substance (“one-side fluorescently labeled single-stranded nucleic acid” in the figure) is prepared (step 102). FIDA measurement is performed on each of these fluorescently labeled nucleic acids, and the fluorescence intensity per molecule is measured (step 103). Let Q1 be the fluorescence intensity of the single-sided fluorescently labeled double-stranded nucleic acid, and Q2 be the fluorescence intensity of the single-sided fluorescently labeled single-stranded nucleic acid. Next, the single-sided fluorescently labeled double-stranded nucleic acid and the test helicase are mixed and reacted (step 104), FIDA measurement is performed on the reaction solution after the reaction, and one molecule of each molecule present in the reaction solution Each hit fluorescence intensity Q3 is measured (step 105). The measurement result is subjected to a two-component analysis with Q1 measured in Step 103 as the first component and Q2 as the second component, and the molecule in the reaction solution is labeled with a single-sided fluorescently labeled double-stranded nucleic acid (“Frac. Q1 ") and one-sided fluorescently labeled single-stranded nucleic acid (" Frac.Q2 "in the figure) (step 106). Thereby, the content ratio of the fluorescently labeled double-stranded nucleic acid in the reaction solution can be calculated (step 107), and the unwinding reaction efficiency of the test helicase can be calculated (step 108).

  In addition, for example, a helicase having a known enzyme activity is used as a control helicase, and the unwinding reaction efficiency obtained by the control helicase is compared with the unwinding reaction efficiency obtained by the test helicase. The enzyme activity of can be evaluated. Specifically, the apparent content ratio of the fluorescently labeled double-stranded nucleic acid in the reaction solution is measured in the same manner as in step (a) except that a control helicase with a known concentration is used instead of the test helicase, The apparent content ratio by this control helicase is compared with the apparent content ratio by the test helicase. For example, when the apparent content ratio of the fluorescently labeled double-stranded nucleic acid by the test helicase is smaller than the apparent content ratio obtained by the control helicase, the remaining double-stranded nucleic acid after the reaction remains under the same reaction conditions. This means that the test helicase has higher unwinding reaction efficiency and higher helicase activity than the control helicase at that concentration.

  The control helicase is not particularly limited as long as it has a known helicase activity such as unwinding reaction efficiency. For example, commercially available purified helicase can be used. In addition, the reaction by the test helicase [step (a)] and the reaction by the control helicase may be performed first, and the operations from irradiation of the reaction solution to excitation light to detection of the fluorescence signal are performed simultaneously. Also good.

  In addition, the control helicase added to the reaction solution was varied in concentration, and the added control was measured by measuring the apparent content of the fluorescently labeled double-stranded nucleic acid in the reaction solution after the reaction with respect to each concentration of reaction solution. The relationship between the concentration of helicase and the apparent content of the fluorescently labeled double-stranded nucleic acid after the reaction can be determined, and the enzyme activity of the test helicase can be evaluated based on the relationship.

  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]
The rewinding reaction efficiency measuring method of the present invention was performed using FCCS as a single molecule fluorescence analysis method.
<Preparation of fluorescently labeled double-stranded nucleic acid>
By performing PCR using a synthetic DNA as a template using a forward primer consisting of 18 bases labeled with rhodamine green at the 5 ′ end and a reverse primer consisting of 19 bases labeled with ATTO633 at the 5 ′ end, 5 ′ A fluorescent-labeled double-stranded nucleic acid (40 base pairs in length), which is an aggregate of a single-stranded nucleic acid labeled with rhodamine green at the end and a single-stranded nucleic acid labeled with ATTO633 at the 5 ′ end, was prepared. Table 1 shows the sequence of each primer.

  PCR was performed by repeating 40 cycles of a thermal cycle consisting of 98 ° C. for 10 seconds, 55 ° C. for 30 seconds, and 72 ° C. for 30 seconds in the reaction solution having the composition shown in Table 2. The obtained PCR product was subjected to electrophoresis using a 15% acrylamide gel, a 40 base pair long band was cut out, and purified using a commercially available purification kit (Mermaid, manufactured by QBiogene) (PAGE purification).

<Preparation of sample series for calibration curve>
The forward primer (Forward primer (Rhodamine Green)) used for the preparation of the above-mentioned fluorescent labeled double-stranded nucleic acid was used as a calibration curve rhodamine green labeled single-stranded nucleic acid for preparing a calibration curve. A reverse primer (Reverse primer (ATTO633)) was used as each strand nucleic acid.
Seven types of calibration were carried out so that the fluorescence-labeled double-stranded nucleic acid prepared above, the rhodamine green-labeled single-stranded nucleic acid for calibration curve, and the ATTO633-labeled single-stranded nucleic acid for calibration curve each had the content ratio shown in Table 3. A sample for wire was prepared. In Table 3, “double strand” is a fluorescently labeled double-stranded nucleic acid, “RhoG single strand” is a rhodamine green labeled single-stranded nucleic acid for calibration curve, and “ATTO single strand” is an ATTO633 labeled single strand for calibration curve. Each nucleic acid means. Each sample solution was prepared by diluting a predetermined amount of nucleic acid with 10 mM Tris-HCl (pH 8.0).

<Creation of calibration curve>
FCCS measurement was performed on each calibration curve sample of the calibration curve sample series prepared above, and the content of the fluorescently labeled double-stranded nucleic acid in the calibration curve sample was measured. The FCCS measurement was performed using a single molecule fluorescence analyzer MF20 (manufactured by Olympus). Rhodamine green was excited by a 488 nm laser (laser power: 100 μW), and ATTO 633 was excited by a 633 nm laser (laser power: 100 μW). The measurement method was measured 5 times for 15 seconds per sample using FCCS.
From the measurement results, the content ratio (DCC) of the fluorescently labeled double-stranded nucleic acid in each calibration curve sample was determined by the above formulas (1) and (2).
5, the vertical axis indicates the apparent content of fluorescently labeled double-stranded nucleic acid (DCC) obtained by FCCS measurement, and the horizontal axis indicates the actual content of fluorescently labeled double-stranded nucleic acid (Frac. G / R-DNA). ) Is a diagram plotting data of each calibration curve sample of the calibration curve sample series. Of these, the least square method or the like was performed on 5 plots (low region) in which the content (Frac.G / R-DNA) of the actual fluorescently labeled double-stranded nucleic acid was 60% or less, and an approximate straight line (in FIG. 5) , “Low area calibration curve”). On the other hand, the least square method or the like is performed on two plots (high region) in which the content ratio (Frac.G / R-DNA) of the actual fluorescently labeled double-stranded nucleic acid exceeds 60%, and an approximate straight line (in FIG. A “high area calibration curve”) was drawn. That is, the calibration curve is a low-area calibration curve when the actual content ratio (Frac.G / R-DNA) of the fluorescently labeled double-stranded nucleic acid is below the boundary between the low region and the high region, and within the range above the boundary. It becomes a high area calibration curve. Note that the intersection of the low region calibration curve and the high region calibration curve is the boundary between the low region and the high region. The slope of each calibration curve is shown below.

<Reaction with test helicase>
The fluorescence-labeled double-stranded nucleic acid prepared above was diluted with a reaction buffer (25 mM Tris-HCl, pH 7.4, 20 mM NaCl, 3 mM MgCl ₂ , 2 mM ATP, 2 mM DTT) so as to be about 5 nM. Six types of reactions in which DNA helicase 2 (manufactured by E. coli, product number: 547-00341, manufactured by Wako Pure Chemical Industries, Ltd.) was added to the prepared fluorescently labeled double-stranded nucleic acid dilution solution so that the final concentration was 0 nM to 66 nM. A solution was prepared. These reaction solutions were reacted at 37 ° C. for 15 minutes. Immediately after the reaction, FCCS measurement was performed, and the content ratio of the fluorescently labeled double-stranded nucleic acid in each reaction solution was measured. The FCCS measurement was performed under the same conditions as in the above <Preparation of calibration curve>. From the measurement results, the content ratio (DCC) of the fluorescently labeled double-stranded nucleic acid in each reaction solution was determined by the above formulas (1) and (2).

  FIG. 6 is a graph showing the apparent content (DCC) of fluorescently labeled double-stranded nucleic acids in the reaction solution measured by FCCS measurement for each concentration of DNA helicase added to the reaction solution. In the reaction solution, a fluorescently labeled double-stranded nucleic acid that has not been subjected to the reaction of helicase, a single-stranded nucleic acid that has been unwound by helicase (a single-stranded nucleic acid labeled with rhodamine green, and a single strand labeled with ATTO633) Strand nucleic acid). The binding ratio DCC calculated by FCCS measurement indicates how much fluorescently labeled double-stranded nucleic acid that has not undergone helicase unwinding is present in the reaction solution.

  Furthermore, from the measured value obtained by the FCCS measurement, the actual content ratio of the fluorescently labeled double-stranded nucleic acid was determined based on the calibration curve shown in FIG. FIG. 7 shows the concentration of the DNA helicase added to the reaction solution based on the actual content ratio of the fluorescence-labeled double-stranded nucleic acid in the reaction solution (remaining ratio of the fluorescence-labeled double-stranded nucleic acid) (Frac.G / R-DNA). ("♦" in the figure). When FCCS measurement is performed on a reaction solution to which helicase has not been added (helicase concentration is 0 nM), the fluorescently labeled double-stranded nucleic acid is present even though only the fluorescently-labeled double-stranded nucleic acid is present in the reaction solution. The apparent content ratio (DCC) is only about 30% (FIG. 6), but when calculated based on the calibration curve, the content ratio of the fluorescently labeled double-stranded nucleic acid is 100%. Thus, by using a calibration curve, it is possible to obtain a more accurate residual rate of fluorescently labeled double-stranded nucleic acid in the reaction solution.

  In addition, a double-stranded DNA (92 base pairs in length) labeled with a radioactive substance is used as a substrate, and a reaction with DNA helicase 2 is performed under the same reaction conditions as in Example 1 (helicase concentration, ATP concentration, reaction temperature, reaction time, etc.). The reaction solution was electrophoresed and the residual rate of double-stranded nucleic acid in the reaction solution was measured experimentally by measuring the radiation dose of a 92 base pair long band (J. Biol. Chem., 262). 2066-2076 (1987)) is shown in accordance with FIG. 7 (“Δ” in the figure). As a result, the FCCS value and the electrophoresis value were approximately the same value. That is, it was found that the unwinding reaction efficiency of helicase can be measured with very high accuracy by using the method for measuring the unwinding reaction efficiency of the present invention.

[Reference Example 1]
A calibration curve was prepared using HiLyte Fluor 488 instead of rhodamine green as a fluorescent substance for labeling the double-stranded nucleic acid.
In place of the forward primer (Forward primer (Rhodamine Green)) used in Example 1, the fluorescently labeled double-stranded nucleic acid used for preparing the calibration curve was replaced by HiLyte Fluor 488 at the 5 ′ end of the oligonucleotide having the same base sequence. It was prepared in the same manner as in <Preparation of fluorescently labeled double-stranded nucleic acid> in Example 1 except that a forward primer [Forward primer (HiLyte488)] consisting of 18 bases was used. In addition, this forward primer [Forward primer (HiLyte488)] was used as a HiLyte488 labeled single-stranded nucleic acid for a calibration curve.

<Preparation of sample series for calibration curve>
Seven types of calibration curves were prepared so that the fluorescence-labeled double-stranded nucleic acid, the calibration curve HiLite 488-labeled single-stranded nucleic acid, and the calibration curve ATTO633-labeled single-stranded nucleic acid had the content ratios shown in Table 3, respectively. A sample was prepared. In Table 3, “double strand” is a fluorescently labeled double stranded nucleic acid, “HiLite single strand” is a HiLite 488 labeled single strand nucleic acid for calibration curve, and “ATTO single strand” is an ATTO 633 labeled single strand nucleic acid for calibration curve. Respectively. Each sample solution was prepared by diluting a predetermined amount of nucleic acid with 10 mM Tris-HCl (pH 8.0).

<Creation of calibration curve>
FCCS measurement was performed on each calibration curve sample of the calibration curve sample series prepared above, and the content of the fluorescently labeled double-stranded nucleic acid in the calibration curve sample was measured. The FCCS measurement was performed using a single molecule fluorescence analyzer MF20 (manufactured by Olympus). HiLyte Fluor 488 was excited by a 488 nm laser (laser power: 100 μW), and ATTO 633 was excited by a 633 nm laser (laser power: 100 μW). The measurement method was measured 5 times for 15 seconds per sample using FCCS.
From the measurement results, the content ratio (DCC) of the fluorescently labeled double-stranded nucleic acid in each calibration curve sample was determined by the above formulas (1) and (2).
8, the vertical axis represents the apparent content (DCC) of fluorescently labeled double-stranded nucleic acid obtained by FCCS measurement, and the horizontal axis represents the actual content of fluorescently labeled double-stranded nucleic acid (Frac. G / R-DNA). ) Is a diagram plotting data of each calibration curve sample of the calibration curve sample series. As a result, as with the calibration curve created in Example 1, there is a calibration curve switching point when the content ratio of the fluorescently labeled double-stranded nucleic acid (Frac. G / R-DNA) is in the range of 60 to 80%. I understood that. Therefore, the least square method or the like is performed on 5 plots (low region) where the content ratio (Frac.G / R-DNA) of the actual fluorescently labeled double-stranded nucleic acid is 60% or less, and an approximate straight line (in FIG. 8, A “low area calibration curve”) was drawn. On the other hand, the least square method or the like is performed on two plots (high region) in which the content ratio (Frac.G / R-DNA) of the actual fluorescently labeled double-stranded nucleic acid exceeds 60%, and an approximate straight line (in FIG. 8, A “high area calibration curve”) was drawn. That is, the calibration curve is a low-area calibration curve when the actual content ratio (Frac.G / R-DNA) of the fluorescently labeled double-stranded nucleic acid is below the boundary between the low region and the high region, and within the range above the boundary. It becomes a high area calibration curve. Note that the intersection of the low region calibration curve and the high region calibration curve is the boundary between the low region and the high region. The slope of each calibration curve is shown below.

  Rhodamine green and HiLyte Fluor 488, which are fluorescent substances for 488 nm, both have a higher brightness of fluorescence per molecule in the single-stranded DNA state than in the double-stranded DNA state. Here, in the analysis by FCCS, if fluorescent molecules having different brightness are mixed, information on brighter molecules tends to be given priority. For this reason, it is presumed that the slope of the calibration curve changes depending on the existence ratio of the fluorescently labeled double-stranded DNA. In particular, when the percentage of single-stranded DNA is large, the DCC (content ratio of double-stranded DNA) calculated by FCCS tends to be smaller than the actual value, and hence the slope of the calibration curve is small. . From Example 1 and Reference Example 1, when a double-stranded nucleic acid used as a substrate is labeled using a fluorescent substance with excitation light of 488 nm, the content of the fluorescent-labeled double-stranded nucleic acid is about 60 to 80%. By the way, it turned out that it is good to change the inclination.

[Reference Example 2]
Using FIDA as a single molecule fluorescence analysis method, the unwinding reaction efficiency of helicase was measured.
<Preparation of single-sided fluorescently labeled double-stranded nucleic acid>
A forward primer (Forward primer (Rhodamine Green)) labeled with rhodamine green at the 5 ′ end used in Example 1 and a reverse primer [Reverse primer (ATTO 633)] used in Example 1 have no nucleotide sequence. An aggregate of a single-stranded nucleic acid whose 5 ′ end is labeled with rhodamine green and an unlabeled single-stranded nucleic acid by performing PCR using a synthetic DNA as a template using a reverse primer comprising a labeled oligonucleotide A one-sided fluorescently labeled double-stranded nucleic acid (40 base pairs long) was prepared. PCR was performed in the same manner as in <Preparation of fluorescently labeled double-stranded nucleic acid> in Example 1.

<Reaction with test helicase>
The one-side fluorescently labeled double-stranded nucleic acid prepared above was diluted with a reaction buffer (25 mM Tris-HCl, pH 7.4, 20 mM NaCl, 3 mM MgCl ₂ , 2 mM ATP, 2 mM DTT) so as to be about 5 nM. . Four kinds of reaction solutions in which DNA helicase 2 (manufactured by E. coli, product number: 547-00341, manufactured by Wako Pure Chemical Industries, Ltd.) was added to this one-side fluorescently labeled double-stranded nucleic acid diluted solution so that the final concentration was 0 nM to 66 nM And these reaction solutions were reacted at 37 ° C. for 15 minutes. The FIDA measurement was performed immediately after the reaction, and the fluorescence intensity per molecule of the fluorescent molecules present in each reaction solution was measured. As a control, the forward primer [Forward primer (Rhodamine Green)] used above was used as a single-sided fluorescently labeled single-stranded nucleic acid, and this was similarly applied to a diluted solution diluted with the reaction buffer so as to be about 5 nM. Then, FIDA measurement was performed to measure the fluorescence intensity Q per molecule. The FIDA measurement was performed using a single molecule fluorescence analyzer MF20 (manufactured by Olympus). Rhodamine green was excited by a 488 nm laser (laser power: 100 μW), and the measurement method was measured five times for 10 seconds per sample using FIDA.

  Fluorescence intensity (12.7 kHz) of double-stranded nucleic acid in a reaction solution having a DNA helicase concentration of 0 nM with respect to the fluorescence intensity Q (kHz) per molecule in the reaction solution of each helicase concentration measured by FIDA measurement Was the first component, and the fluorescence intensity (19 kHz) of the unwound single-stranded nucleic acid was the second component, a two-component analysis was performed, and the ratio of the molecules of each fluorescence intensity was calculated. FIG. 9 shows the content ratio (Q = 12.7) of the one-sided fluorescently labeled double-stranded nucleic acid in the reaction solution of each helicase concentration as a result of performing two-component analysis on the fluorescence intensity measured by FIDA measurement. The content ratio (Q = 19) of the single-sided fluorescently labeled single-stranded nucleic acid is shown. From this result, it was found that the unwinding reaction efficiency when the DNA helicase concentration was 66 nM was about 80%, about 50% when 26 nM, and about 15% when 13 nM.

  FIG. 10 shows the unwinding reaction efficiency (%) at each helicase concentration measured in the same manner as the electrophoresis method shown in FIG. 7 (“Δ” in the figure). Comparing FIG. 9 and FIG. 10, it was found that the unwinding reaction efficiencies of the two at the respective helicase concentrations were almost the same, and the helicase activity could also be measured by using FIDA analysis.

  By using the method for measuring the unwinding reaction efficiency of the present invention, the unwinding reaction efficiency of the helicase with respect to the double-stranded nucleic acid can be accurately and quantitatively measured without performing complicated work. In addition, it can be used in the field of development of nucleic acid medicines utilizing helicase activity.

Claims (4)

A method for measuring the unwinding reaction efficiency of a helicase to a double-stranded nucleic acid,
(A) a first fluorescently labeled single-stranded nucleic acid labeled with a first fluorescent substance, and a second fluorescently labeled single-stranded nucleic acid labeled with a second fluorescent substance having a spectral characteristic different from that of the first fluorescent substance; Light having a wavelength capable of exciting the first fluorescent substance and the second fluorescent substance in the reaction solution after adding helicase to the reaction solution containing the fluorescently labeled double-stranded nucleic acid that is an aggregate of The fluorescence emitted from each fluorescent substance is detected as a fluorescence signal, and the detected fluorescence signal is analyzed by a single molecule fluorescence analysis method to detect the fluorescence in the reaction solution. Measuring the apparent content of labeled double-stranded nucleic acid;
(B) An equal amount of a first fluorescent-labeled single-stranded nucleic acid for a calibration curve labeled with the first fluorescent substance and a second fluorescent-labeled single-stranded nucleic acid for a calibration curve labeled with the second fluorescent substance And a calibration curve fluorescent labeled double-stranded nucleic acid labeled with the first fluorescent substance and the second fluorescent substance, the calibration curve first fluorescent labeled single-stranded nucleic acid, and the calibration curve second fluorescent label. Preparing a calibration curve sample series in which the content ratio of the fluorescence-labeled double-stranded nucleic acid for calibration curve is different from the total with the single-stranded nucleic acid;
(C) Light of a wavelength that can excite the first fluorescent substance and light of a wavelength that can excite the second fluorescent substance to each of the calibration curve samples of the calibration curve sample series prepared in the step (b). , Respectively, and the fluorescence emitted from each fluorescent substance is detected as a fluorescence signal, the detected fluorescence signal is analyzed by a single molecule fluorescence analysis method, and the calibration curve fluorescent label 2 in the calibration curve sample is analyzed. A step of measuring an apparent content ratio (molar ratio) of the double-stranded nucleic acid;
(D) The apparent content ratio (molar ratio) of the fluorescent labeled double-stranded nucleic acid for the calibration curve of each calibration curve sample measured in the step (c), and two actual fluorescent labels for the calibration curve in the measurement sample Creating an approximate line of the relationship with the content of the chain nucleic acid;
(E) Based on the approximate line created in the step (d) and the apparent content ratio (molar ratio) of the fluorescently labeled double-stranded nucleic acid in the reaction solution measured in the step (a), the winding of the helicase Calculating the return reaction efficiency;
A method for measuring the unwinding reaction efficiency of a helicase, comprising:   The method for measuring the unwinding reaction efficiency of helicase according to claim 1, wherein the single molecule fluorescence analysis method is fluorescence cross-correlation spectroscopy. The fluorescent material is a fluorescent material that is excited by excitation light having a wavelength of 488 nm,
The approximate line created in the step (d) is composed of an approximate line in the low region of the actual content of the fluorescent labeled double-stranded nucleic acid for the calibration curve in the measurement sample and an approximate line in the high region of the content rate. ,
The method for measuring the unwinding reaction efficiency of a helicase according to claim 1 or 2, wherein a boundary between the low region and the high region is in a range of 60 to 80%.   The method for measuring the unwinding reaction efficiency of a helicase according to claim 3, wherein a boundary between the low region and the high region is in a range of 60 to 70%.

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