JP5586864B2 - Method for measuring helicase activity - Google Patents

Description

  The present invention relates to a method and a reagent for simply measuring the activity of a helicase that is an enzyme that unwinds a double-stranded nucleic acid such as double-stranded DNA or double-stranded RNA into a single strand.

  Proteins having the activity of unwinding a double-stranded nucleic acid such as double-stranded DNA or double-stranded RNA into a single strand are generally referred to as helicases. Among helicases, those that act on double-stranded DNA are conveniently called DNA helicases, and those that act on double-stranded RNA are called RNA helicases for convenience. Recent research has shown that many RNA helicases also act on double-stranded DNA. Thus, it has become clear that single-stranded DNA is produced. For this reason, double-stranded DNA is often used as a substrate for RNA helicase activity measurement.

  Helicases are rich in diversity and have a wide variety of biological roles, so they have been attracting attention as biomedical research targets in recent years. At the same time, they are also regarded as important for developing drugs against specific pathogens such as viruses. Yes. For example, RNA helicases derived from RNA viruses are attracting attention as targets for antiviral agents because they differ greatly from mammalian helicases. For example, Patent Documents 1 and 2 disclose helicase activity measurement methods and drug screening methods intended for the above-mentioned use. Helicases are also used in DNA amplification techniques in vitro, and several types of helicases, such as E. coli-derived uvrD helicase and T7 gene 4 helicase, are disclosed in Patent Document 3 as helicases used for the above purpose.

As a method for measuring helicase activity, radioisotopes such as ³² P are often used regardless of whether the substrate is double-stranded DNA or double-stranded RNA (Patent Documents 1 and 2, Non-Patent Documents 1 and 2). 2) or a substrate labeled with a fluorescent dye (Patent Documents 4 and 5, Non-Patent Documents 3 and 4) is used. Further, as a special method, a method using surface plasmon resonance (Patent Document 6), a method using a reaction between a biotin-labeled nucleic acid and streptavidin-labeled agarose beads (Patent Document 2) and the like are disclosed. However, the above-described helicase activity measurement method has a problem that the reaction substrate is expensive or chemically unstable, and the apparatus necessary for the measurement is special.

US Pat. No. 5,705,344 Special Table 2000-508521 JP 2006-500028 Gazette JP 2005-80535 A JP 2008-99619 A Special Table 2002-540800

Tai, C.I. Et al., Journal of Virology, 70 (12), 8477-8484, 1996. Tanner, J .; A. The Journal of Biological Chemistry, 278 (41), 39578-39582, 2003. Houston, P.M. Proceedings of National Academy of Sciences USA, 91, 5471-5474, 1994. Raney, K.M. D. Et al., Proceedings of National Academy of Sciences USA, 91, 6644-6648, 1994. Seybert, A.M. Et al., RNA, 6, 1056-1068, 2000 Seerebrov, V.M. Et al., Nature, 430, 476-480, 2004. Denison, M.M. R. Et al., Journal of Virology, 73 (8), 6862-6871, 1999. Chouljenko, V.M. N. Et al., Journal of General Virology, 82, 2927-2933, 2001.

  The problem to be solved by the present invention is to provide a method and a reagent for measuring the enzyme activity of helicase easily and inexpensively.

  As a result of intensive studies to solve the above problems, the present inventor has made use of the fact that the single-stranded nucleic acid generated by the helicase reaction forms a double strand again in the reaction solution, and thus the helicase reaction substrate and the helicase. By detecting the reaction product separately, it was found that helicase activity can be measured easily and inexpensively, and the present invention has been completed.

That is, the present invention includes the following inventions:
The first invention is
A partially double-stranded nucleic acid (A) composed of a single-stranded nucleic acid (A-1) and a single-stranded nucleic acid (A-2), a single-stranded nucleic acid (B), and a helicase,
A method for measuring helicase activity, comprising measuring a double-stranded nucleic acid or a partially double-stranded nucleic acid (C) comprising a single-stranded nucleic acid (A-2) and a single-stranded nucleic acid (B). ,
The nucleic acid (A-1) comprises a sequence containing a sequence (helicase recognition sequence) necessary for an enzymatic reaction with helicase at the 5 ′ end and / or 3 ′ end,
The nucleic acid (A-2) comprises a sequence complementary to a portion other than the helicase recognition sequence in the nucleic acid (A-1),
The nucleic acid (B) comprises a sequence complementary to a part of the nucleic acid (A-2), or a sequence completely complementary to the nucleic acid (A-2), and (A-2 In the above method, the nucleic acid comprises a sequence that does not contain a helicase recognition sequence when base-paired with a nucleic acid.

  The second invention is the measuring method according to the first invention, characterized in that the nucleic acids (A-1), (A-2) and (B) are unlabeled nucleic acids.

  A third invention is the measurement method according to the first or second invention, wherein the measurement of the nucleic acid of (C) is performed using electrophoresis.

  The fourth invention is characterized in that the helicase is a helicase derived from a virus of the Coronaviridae or Flaviviridae family, or any of these mutants, partial deletions, and fusions. It is the measuring method as described in three inventions.

The fifth invention is a helicase activity comprising a single-stranded nucleic acid (A-1), a single-stranded nucleic acid (A-2), a single-stranded nucleic acid (B), a nucleotide triphosphate, and a divalent metal salt. Measuring reagent,
The nucleic acid (A-1) comprises a sequence containing a sequence (helicase recognition sequence) necessary for an enzymatic reaction with helicase at the 5 ′ end and / or 3 ′ end,
The nucleic acid (A-2) comprises a sequence complementary to a portion other than the helicase recognition sequence in the nucleic acid (A-1),
The nucleic acid (B) comprises a sequence complementary to a part of the nucleic acid (A-2), or a sequence completely complementary to the nucleic acid (A-2), and (A-2 The reagent is composed of a sequence that does not contain a helicase recognition sequence when base-paired with the nucleic acid of (1).

  Hereinafter, the present invention will be described in detail.

The helicase in the present invention refers to a protein having an activity of unwinding a double-stranded nucleic acid such as double-stranded DNA or double-stranded RNA into a single strand. For convenience, those that act on double-stranded DNA are called DNA helicases, and those that act on double-stranded RNA are called RNA helicases, but the selectivity for both substrates varies greatly depending on the enzyme and acts almost equally on both substrates. There are many enzymes. Here, except those that have been established as proper nouns, they are simply referred to as helicases without distinction. Helicases are often associated with other enzyme activities, of which typical is NTPase activity. Since the hydrolysis energy of NTP (nucleotide triphosphate) is required for the expression of helicase activity, NTPase activity is observed in all helicases. In addition, ATP (adenosine triphosphate) is often used most efficiently among NTPs. In addition, helicases, like many other nucleic acid-related enzymes, require divalent metal ions such as Mg ²⁺ and Mn ²⁺ for their activity.

  The partial double-stranded nucleic acid in the present invention is a 5 ′ end and / or 3 ′ end side of a continuous nucleotide region (double-stranded region) that forms a stable inter-strand hydrogen bond near room temperature and under low ionic strength. In addition, it refers to a nucleic acid having a continuous nucleotide region (single-stranded region) that does not form a stable hydrogen bond near room temperature and low ionic strength.

The unlabeled nucleic acid in the present invention refers to a nucleic acid to which a radioisotope such as ³² P, a fluorescent dye such as fluorescein, or a labeled compound that enables a special intermolecular interaction such as biotin is not added. Say.

  The sequence (helicase recognition sequence) necessary for the enzymatic reaction by helicase in the present invention refers to a sequence necessary for the unwinding reaction of a specific helicase. The length of the recognition sequence varies depending on each helicase. For example, in the case of human coronavirus 229E-derived helicase, a single-stranded region having a length of about 10 bases is required at the 5 ′ end (Non-patent Document 5). In the case of viruses, conversely, a single-stranded region of about 20 bases is required on the 3 ′ end side (in Non-Patent Document 6, a partial double-stranded DNA having a single-stranded region of 18 bases is prepared, and an enzyme reaction Used).

  Next, the method for measuring helicase activity of the present invention will be described.

  The nucleic acid sequences of single-stranded nucleic acid (A-1), single-stranded nucleic acid (A-2), and single-stranded nucleic acid (B) used in the present invention are extremely diverse, and specific nucleic acids It is not limited to the sequence. Moreover, although there is no restriction | limiting about base length, the partial double stranded nucleic acid (A) formed from the nucleic acid of said (A-1) and (A-2), and said (A-2) and (B) The partial double-stranded nucleic acid (C) formed from the nucleic acid is preferably a nucleic acid having a double-stranded region of 20 to 1000 base pairs and a single-stranded region of 10 to 100 bases. The double-stranded nucleic acid formed from the nucleic acids (A-2) and (B) is preferably a nucleic acid having 20 to 1000 base pairs.

  The nucleic acids (A-1), (A-2), and (B) may be labeled with a labeling compound that enables a special intermolecular interaction such as a radioisotope, a fluorescent dye, or biotin. However, the measurement method of the present invention can be applied even to an unlabeled nucleic acid. Moreover, since unlabeled nucleic acid can be synthesized easily and inexpensively compared with labeled nucleic acid, it can be said to be a preferred embodiment as a nucleic acid used in the measurement method of the present invention.

  A partially double-stranded nucleic acid can be easily prepared by mixing equimolar amounts of two types of single-stranded nucleic acids that form stable hydrogen bonds between strands and base-pairing (annealing). In order to perform the annealing operation more efficiently, an operation such as heat treatment and subsequent slow cooling or addition of an appropriate inorganic salt may be used in combination. In order to increase the yield of the intended partial double-stranded nucleic acid, it is preferable that each single-stranded nucleic acid has less self-complementary sequences. In order to design the preferred sequences, GENETYX (trade name) (Genetics) A sequence analysis program such as

  Nucleic acids include single and double stranded DNA and RNA. Double-stranded nucleic acids include those in which the pair of strands is DNA-DNA, RNA-RNA, and DNA-RNA. Unlabeled DNA can be obtained at low cost by chemical synthesis using a standard nucleic acid synthesizer. In many cases, the bases of the constituting nucleotides are four types of A (adenine), G (guanine), T (thymine), and C (cytosine), but a derivative such as I (inosine) may be included. . Unlabeled DNA can also be obtained by treating plasmid DNA grown using gene recombination technology with an appropriate restriction enzyme. In this case, the bases of the constituting nucleotides are often four types of A (adenine), G (guanine), T (thymine), and C (cytosine), but may contain a base that is methylated by the host cell. . The DNA fragment prepared by the above method is basically double-stranded (may contain a short single-stranded region at the end), but is suitable for a method using a DNA polymerase from the DNA fragment or a heat denaturation operation. Single-stranded DNA can be prepared by using a chromatography operation in combination. On the other hand, unlabeled RNA can be enzymatically synthesized using double-stranded DNA as a template and RNA polymerase such as T7 RNA polymerase or SP6 RNA polymerase. In this case, the bases of the nucleotides to be constructed are in most cases four types: A (adenine), G (guanine), U (uracil) and C (cytosine). Single-stranded unlabeled nucleic acid (DNA, RNA) synthesized by the above method can be used as it is after purification by ordinary chromatography or electrophoresis operation, or used as a partial double-stranded nucleic acid by the above-mentioned annealing operation. You can also

  In addition to single-stranded nucleic acid (A-1), single-stranded nucleic acid (A-2) and single-stranded nucleic acid (B), the solution for measuring helicase activity includes NTP (preferably ATP), bivalent It is necessary that each metal salt (preferably magnesium salt) is contained in an appropriate concentration. The composition may be optimized according to the helicase to be measured, or may be fixed to a general-purpose composition. In addition, a reagent having a buffering capacity near pH 7, a mild reducing agent such as DTT (dithiothreitol), an enzyme stabilizer such as BSA (bovine serum albumin) and glycerol, Tween 80 (trade name), etc. A mild surfactant or the like may be added as appropriate.

  The temperature of the enzyme reaction by helicase is not particularly limited, and a temperature suitable for the enzyme reaction may be selected between 0 ° C. and 80 ° C. Generally, in the case of thermostable helicase, the speed of the enzyme reaction by helicase is increased. In order to reduce the secondary structure of the nucleic acid, a higher reaction temperature is selected.

  As a method for measuring the nucleic acid of the double-stranded nucleic acid or partial double-stranded nucleic acid (C) generated by the enzymatic reaction with helicase, the nucleic acid of (C) is separated from the nucleic acid of (A) and (B). Any method can be used as long as it can detect at least the nucleic acid (C). As a method for separating the nucleic acid (C) from the nucleic acids (A) and (B), a liquid using a column packed with a separating agent such as TSKgel DNA-NPR (trade name) (manufactured by Tosoh Corporation) Examples of the separation method using chromatography and the separation method using gel electrophoresis such as polyacrylamide gel electrophoresis and agarose gel electrophoresis are examples. However, the separation method using gel electrophoresis does not require an expensive apparatus and is easy to operate. This is preferable. In particular, when the base lengths of the nucleic acids (A), (B), and (C) are short (for example, 200 base pairs or less), polyacrylamide gel electrophoresis that allows easy separation of short nucleic acids is preferable. The separation method using capillary electrophoresis also requires an expensive apparatus such as P / ACE system (trade name) (Beckman Coulter) or ABI PRISM 3100 (trade name) (Applied Biosystems). This can be said to be an embodiment of a preferable separation method in that precise separation can be performed.

  As a method for detecting the nucleic acid (C), when the nucleic acid (C) is pre-labeled, an apparatus that specifically detects the labeled radioisotope, fluorescent dye, or labeled compound is used. What is necessary is just to detect. In addition, when the nucleic acid (C) is an unlabeled nucleic acid, the nucleic acid (C) is converted with a fluorescent dye such as ethidium bromide or SYBR Green (trade name) (manufactured by Takara Bio Inc.) before and after the separation operation. What is necessary is just to stain and detect. In the detection operation, the nucleic acid of (C) can be quantified by using a nucleic acid with a known concentration as a standard substance.

  When the nucleic acids (A) and (B) are unlabeled nucleic acids and the reaction product separated by gel electrophoresis is stained with a fluorescent dye such as ethidium bromide, the substrate is about 0.1 to 1 pmol / μL. What is necessary is just to make it adjust to a density | concentration and to make it react. However, since the single-stranded nucleic acid, which is the primary product of the helicase reaction, quickly returns to double-stranded at a concentration in the vicinity, it may be observed that the reaction hardly seems to proceed. In order to avoid the above problem, it is preferable to add the nucleic acid (B) in an equimolar amount or more, preferably 5 molar ratio or more with respect to the nucleic acid (A). By doing so, the single-stranded nucleic acid (A-2) produced from the nucleic acid (A) by the enzymatic reaction of helicase forms a base pair with the (A-1) again, and the (A) The probability of base pairing with the nucleic acid of (B) to become the nucleic acid of (C) is higher than returning to the nucleic acid of Since the nucleic acid of (C) does not have a sequence (helicase recognition sequence) necessary for the enzymatic reaction by helicase, the nucleic acid of (C) is likely to accumulate as a result, and the nucleic acid detection of (C) is also easy. It becomes.

  The type of helicase in the present invention is not particularly limited, but a preferred embodiment of the present invention is to use a helicase that requires a single-stranded region on the 5 'end side or 3' end side for initiation of the reaction. Many of the helicases are known. Among them, helicases derived from viruses belonging to the aforementioned Coronaviridae group are helicases derived from viruses belonging to the Flaviviridae family as a group requiring a single-stranded nucleic acid at the 5 ′ end side. Has been analyzed in detail as a group requiring a single-stranded nucleic acid on the 3 ′ end side, and thus is preferred as the target helicase in the measurement method of the present invention. The former includes human coronavirus (HCoV) and SARS coronavirus (SARS-CoV), and the method of the present invention is preferable for searching for drugs against these viruses. In addition, mouse hepatitis virus (MHV), bovine coronavirus (BCoV), and the like are not pathogenic to humans, but are important viruses from the viewpoint of management of laboratory animals and livestock animals, and the genome of the virus is human coronavirus. Since the origin and the related helicase are encoded (Non-patent Documents 7 and 8), the helicase is also a preferable application example of the present invention. On the other hand, the latter includes hepatitis C virus (HCV) and yellow fever virus (YFV), and the method of the present invention is also preferable for searching for drugs against these viruses.

Virus-derived helicase is not a constituent of virus particles, and generally, the intracellular content of helicase is very small, so it takes a lot of labor to collect naturally occurring enzymes. Therefore, it is generally obtained using a genetic recombination technique. Genetically modified enzymes are usually
(1) Amplification of enzyme gene,
(2) Integration of the amplified gene into a cloning vector,
(3) transformation of host cells,
(4) culture of transformed cells,
(5) Enzyme purification from culture medium or cells,
However, for the purpose of improving the productivity of the enzyme and / or facilitating purification, a product having a partially different amino acid sequence from the natural enzyme (mutant), the natural enzyme In many cases, it is produced as a product in which a part of the amino acid sequence is deleted (partial deletion product) or a product fused with another functional protein or peptide (fusion product). The method for measuring helicase according to the present invention has the enzyme activity of helicase, and recognizes helicase in the nucleic acid of (A-1), even if it is the mutant, the partial deletion, or the fusion. If the sequence can be recognized, the method for measuring helicase of the present invention can be applied.

  In addition, since handling of viruses often requires special equipment and operational precautions for the purpose of preventing infection to humans and animals, it is necessary to use already cloned genes without using viruses or virus-infected cells. It is preferable to amplify a virus-derived enzyme gene. If a cloned gene is not available, the enzyme gene can be fully synthesized based on the nucleotide sequence information. The specific method of the latter will be described in Example 1 below.

  According to the present invention, helicase activity can be measured easily and inexpensively, and a chemically stable and inexpensive reagent for measuring helicase activity can also be provided. In addition, according to the measurement method and reagent, human coronavirus (HCoV), SARS coronavirus (SARS-CoV), mouse hepatitis virus (MHV), bovine coronavirus (BCoV), hepatitis C virus (having helicase activity) It is possible to efficiently search for drugs against viruses such as HCV) and yellow fever virus (YFV).

Measurement result of helicase activity (Example 6). The conceptual diagram of reaction in Example 6. FIG. Measurement results of helicase activity (Example 7). Measurement result of helicase activity (Example 8). Measurement result of helicase activity (Example 9). The conceptual diagram of reaction in Example 9. FIG.

  EXAMPLES Hereinafter, although an Example and a reference example demonstrate this invention in detail, this invention is not limited to these.

Example 1 Synthesis of mouse hepatitis virus (MHV) helicase gene The mouse hepatitis virus (MHV) helicase gene was artificially synthesized by the method described below.
(1) 30 pmol each of oligonucleotides consisting of the sequences shown in SEQ ID NOs: 1 and 12, 1.5 pmol each of oligonucleotides consisting of the sequences shown in SEQ ID NOs: 2 to 11, and PrimeSTAR HS DNA polymerase (trade name) (Takara) 50 μL of a PCR reaction solution containing Bio (manufactured by Bio Inc.) was prepared, and a PCR reaction was performed to obtain a PCR product of 623 base pairs.
(2) 30 pmol each of oligonucleotides having the sequences shown in SEQ ID NOs: 13 and 24, 1.5 pmol each of oligonucleotides having the sequences shown in SEQ ID NOs: 14 to 23, and PrimeSTAR HS DNA polymerase (trade name) (Takara) 50 μL of a PCR reaction solution containing Bio (manufactured by Bio Inc.) was prepared, and a PCR reaction was performed to obtain a 624 base pair PCR product.
(3) 30 pmol each of oligonucleotides consisting of the sequences shown in SEQ ID NOs: 25 and 36, 1.5 pmol each of oligonucleotides consisting of the sequences shown in SEQ ID NOs: 26 to 35, and PrimeSTAR HS DNA polymerase (trade name) (Takara) 50 μL of a PCR reaction solution containing Bio (manufactured by Bio Inc.) was prepared, and a PCR reaction was performed to obtain a PCR product of 623 base pairs.
(4) After purifying the PCR product prepared in the above (1) to (3) using agarose gel electrophoresis, each recovered PCR product is about 1 pmol each, and an oligonucleotide having the sequence described in SEQ ID NOs: 1 and 36 PCR reaction solution containing 30 pmol of each and PrimeSTAR HS DNA polymerase (trade name) (manufactured by Takara Bio Inc.) was prepared, and a PCR reaction was performed to obtain a PCR product of 1830 base pairs.

Example 2 Preparation of MHV helicase-producing E. coli Recombinant E. coli expressing the helicase was prepared from the MHV helicase gene prepared in Example 1.
(1) The 1830 base pair PCR product prepared in Example 1 was treated with restriction enzymes EcoRI and HindIII to make both ends of the fragment cohesive ends of EcoRI and HindIII, respectively, and then treated with EcoRI and HindIII. Ligation was performed using the vector pTrc99A (GenBank No. U13872) and T4 DNA ligase.
(2) Recombinant E. coli JM109 / pM HV_HEL3 was obtained by transforming competent cells of E. coli JM109 (manufactured by Takara Bio Inc.) by the heat shock method using the ligated plasmid.
(3) As a result of culturing the recombinant E. coli and analyzing the base sequence of the plasmid extracted from the cells, it was confirmed that the sequence as designed was included. The result of nucleotide sequence analysis is shown in SEQ ID NO: 37 together with the amino acid sequence of the translation product.

Example 3 Preparation of MHV helicase preparation MHV helicase used in the examples was prepared from the recombinant E. coli prepared in Example 2.
(1) 1 L of 2 × YT medium containing 50 μg / mL of carbenicillin (composition: 16 g / L Bacto Trypton, 10 g / L Yeast Extract, 5 g / L NaCl, the recombinant E. coli JM109 / pMHV_HEL3 obtained in Example 2) After adjusting the pH to 7 with NaOH at 37 ° C., 0.5 mM IPTG was added when the OD ₆₀₀ reached about 1, and the culture was further continued at 22 ° C. for 16 hours. The culture was ice-cooled, and the subsequent operations were performed under 4 ° C cooling.
(2) The culture was centrifuged (6000 rpm, 20 minutes) to remove the culture supernatant. The bacterial pellet is suspended in 90 mL of Lysis buffer (composition: 20 mM HEPES, 500 mM NaCl, 1 mM EDTA, pH 7.5), then centrifuged (8000 rpm, 30 minutes) to remove the supernatant, and the bacterial pellet is protease inhibitor. The suspension was resuspended in 60 mL of Lysis buffer solution (trade name) (manufactured by Roche Diagnostics) (1 tablet dissolved in 50 mL of Lysis buffer solution).
(3) The suspension was crushed with an ultrasonic crusher, and centrifuged (15000 rpm, 30 minutes) to obtain a supernatant. A saturated ammonium sulfate solution was added to the supernatant to a saturation concentration of 25%, left to stand for 30 minutes, and then centrifuged (8000 rpm, 20 minutes) to obtain a supernatant. A saturated ammonium sulfate solution was added to the obtained supernatant to a saturation concentration of 45%, left to stand for 30 minutes, and then centrifuged (8000 rpm, 20 minutes) to remove the supernatant and obtain a precipitate. The obtained precipitate was dissolved in 5 mL of Lysis buffer containing the protease inhibitor cocktail and dialyzed against 2 L of Lysis buffer for 4 hours.
(4) 3 times volume of buffer I (composition: 20 mM HEPES, 0.6 M ammonium sulfate, 1 mM EDTA, pH 7.5) was added to the protein solution after dialysis, and this was equilibrated with buffer I 5 mL of HiTrap Phenyl. (Product name) (GE Healthcare Bioscience) applied to the column, and 50 mL of buffer solution was passed through to wash the column. Next, the column was washed in the same manner using 50 ml of buffer II (composition: 20 mM HEPES, 0.3 M ammonium sulfate, 1 mM EDTA, pH 7.5). Thereafter, buffer solution III (composition: 20 mM HEPES, 1 mM EDTA, pH 7.5) was passed through to separate 0.5 mL of the eluted protein, thereby obtaining a partially purified MHV helicase preparation.

Example 4 Preparation of nucleic acid for measuring helicase activity (1)
(1) An oligonucleotide consisting of the sequence shown in SEQ ID NO: 38 was designed as a single-stranded nucleic acid (A-1), and an oligonucleotide consisting of the sequence shown in SEQ ID NO: 39 was designed as a single-stranded nucleic acid (A-2).
(2) An aqueous solution having each composition shown in Table 1 was prepared, heat-treated in DNA Thermal Cycler (manufactured by PERKIN ELMER CETUS) at 95 ° C. for 2 minutes, and then annealed by stopping the heat source of the apparatus and gradually cooling. It was.

（３）アニーリング後、１２％ポリアクリルアミドゲル電気泳動により部分二本鎖ＤＮＡを分離後、ゲルからの電気溶出とエタノール沈殿によって精製することにより、配列番号３８及び３９に記載の配列からなるオリゴヌクレオチドが塩基対形成した、５ｐｍｏｌ／μＬの部分二本鎖核酸（Ａ）水溶液（以降部分二本鎖ＤＮＡ−１と呼ぶ）４００μＬを得た。

(3) After annealing, the partial double-stranded DNA is separated by 12% polyacrylamide gel electrophoresis, and then purified by electroelution from the gel and ethanol precipitation, whereby the oligonucleotide consisting of the sequences of SEQ ID NOs: 38 and 39 400 μL of 5 pmol / μL partially double-stranded nucleic acid (A) aqueous solution (hereinafter referred to as “partial double-stranded DNA-1”) was obtained.

Example 5 Preparation of nucleic acid for measuring helicase activity (Part 2)
(1) An oligonucleotide consisting of the sequence shown in SEQ ID NO: 41 as a single-stranded nucleic acid (A-1) and an oligonucleotide consisting of the sequence shown in SEQ ID NO: 42 as a single-stranded nucleic acid (A-2) were designed.
(2) An aqueous solution having each composition shown in Table 2 was prepared, heat-treated in DNA Thermal Cycler (manufactured by PERKIN ELMER CETUS) at 95 ° C. for 2 minutes, and then annealed by stopping the heat source of the apparatus and gradually cooling. It was.

（３）アニーリング後、１２％ポリアクリルアミドゲル電気泳動により部分二本鎖ＤＮＡを分離後、ゲルからの電気溶出とエタノール沈殿によって精製することにより、配列番号４１及び４２に記載の配列からなるオリゴヌクレオチドが塩基対形成した、５ｐｍｏｌ／μＬの部分二本鎖核酸（Ａ）水溶液（以降部分二本鎖ＤＮＡ−２と呼ぶ）を４００μＬ得た。

(3) After annealing, the partially double-stranded DNA is separated by 12% polyacrylamide gel electrophoresis, and then purified by electroelution from the gel and ethanol precipitation, whereby the oligonucleotide consisting of the sequences of SEQ ID NOs: 41 and 42 400 μL of 5 pmol / μL partially double-stranded nucleic acid (A) aqueous solution (hereinafter referred to as partially double-stranded DNA-2) was obtained.

Example 6 Measurement of helicase activity (1)
The helicase activity of each fraction obtained in Example 3 was measured by the following method.
(1) A reaction solution having the composition shown in Table 3 was prepared and reacted at 30 ° C. for 30 minutes. As the single-stranded nucleic acid (B), an oligonucleotide having the sequence set forth in SEQ ID NO: 40 is designed and used.

（２）反応後、反応停止溶液（組成：１％ ＳＤＳ、１００ｍＭ ＥＤＴＡ、ｐＨ８．０）を１０μＬ添加し、うち１０μＬを１２％ポリアクリルアミドゲル電気泳動により分析した。

(2) After the reaction, 10 μL of a reaction stop solution (composition: 1% SDS, 100 mM EDTA, pH 8.0) was added, 10 μL of which was analyzed by 12% polyacrylamide gel electrophoresis.

  The result of staining the gel after electrophoresis with ethidium bromide is shown in FIG. In FIG. 1, F number in each lane indicates the result when each fraction obtained in Example 3 is used as an enzyme solution, N indicates the result when water is added instead of the enzyme solution, and M indicates a molecular weight marker. Respectively. Further, (A) is obtained by partial double-stranded DNA-1 obtained in Example 4, (B) is obtained by oligonucleotide comprising the sequence described in SEQ ID NO: 40, and (C) is obtained by enzymatic reaction with helicase. The band corresponding to the partial double-stranded nucleic acid in which the oligonucleotide having the sequence set forth in SEQ ID NO: 39 and the oligonucleotide having the sequence set forth in SEQ ID NO: 40 are base-paired is shown. In addition, the conceptual diagram of reaction in a present Example is shown in FIG. 2 (Here, (A), (B) and (C) represent the same nucleic acid as FIG. 1). From FIG. 1, it was found that helicase activity was observed in each fraction, and in particular, the enzyme activities of fractions 5 to 7 were high.

Example 7 Measurement of helicase activity (2)
(1) Using the fraction 6 in Example 3 (F6 in FIG. 1) as an enzyme solution, a reaction solution having the composition shown in Table 3 was prepared, and then at 30 ° C. for 0 minutes, 5 minutes, 10 minutes, 20 minutes, 30 The reaction was performed for 40 minutes and 60 minutes, respectively.
(2) After the reaction, 10 μL of a reaction stop solution (composition: 1% SDS, 100 mM EDTA, pH 8.0) was added, 10 μL of which was analyzed by 12% polyacrylamide gel electrophoresis.

  The result of staining the gel after electrophoresis with ethidium bromide is shown in FIG. In FIG. 3, the numbers in each lane indicate the time (minutes) of reaction with the enzyme solution, M indicates the molecular weight marker, and (A), (B), and (C) indicate the same nucleic acid bands as in FIG. Since the reaction product increased with time, it was found that the enzyme reaction by the helicase obtained this time lasted at least 60 minutes after the start of the reaction.

Example 8 Measurement of helicase activity (part 3)
(1) Fraction 7 in Example 3 (F7 in FIG. 1) was used as an enzyme solution, and H ₂ O was added instead of the oligonucleotide consisting of the sequence shown in SEQ ID NO: 40 in the composition shown in Table 3. After preparing the reaction solution having the composition, the reaction solution was reacted at 30 ° C. for 30 minutes.
(2) After the reaction, 10 μL of a reaction stop solution (composition: 1% SDS, 100 mM EDTA, pH 8.0) was added, and 10 μL thereof was analyzed by 12% polyacrylamide gel electrophoresis together with the oligonucleotides of SEQ ID NOs: 38 and 39. .

  FIG. 4 shows the result of staining the gel after electrophoresis with ethidium bromide. In FIG. 4, M represents a molecular weight marker, and (A), (B), and (C) represent the same nucleic acid bands as in FIG. When the oligonucleotide (single-stranded nucleic acid (B)) having the sequence shown in SEQ ID NO: 40 is not present in the reaction solution (lane 2 in FIG. 4), the single-stranded nucleic acid (A-1) produced by the enzymatic reaction And (A-2) (an oligonucleotide consisting of the sequences described in SEQ ID NOs: 38 and 39) rapidly forms a partially double-stranded nucleic acid (A), and thus it is found that detection of enzyme activity is extremely difficult.

Example 9 Measurement of helicase activity (4)
(1) Using the fraction 6 (F6 in FIG. 1) shown in Example 3 as an enzyme solution, a reaction solution having the composition shown in Table 4 was prepared, and then at 30 ° C. for 0 minutes, 10 minutes, 20 minutes, and 30 minutes.・ Reacted for 60 minutes. As the single-stranded nucleic acid (B), an oligonucleotide having the sequence set forth in SEQ ID NO: 43 is designed and used.

（２）反応後、反応停止溶液（組成：１％ ＳＤＳ、１００ｍＭ ＥＤＴＡ、ｐＨ８．０）を１０μＬ添加し、うち１０μＬを１２％ポリアクリルアミドゲル電気泳動により分析した。

(2) After the reaction, 10 μL of a reaction stop solution (composition: 1% SDS, 100 mM EDTA, pH 8.0) was added, 10 μL of which was analyzed by 12% polyacrylamide gel electrophoresis.

  The result of staining the gel after electrophoresis with ethidium bromide is shown in FIG. In FIG. 5, the numbers in each lane represent the time (minutes) of reaction with the enzyme solution, and M represents a molecular weight marker. Further, (A) is obtained by partial double-stranded DNA-2 obtained in Example 5, (B) is obtained by oligonucleotide having the sequence described in SEQ ID NO: 43, and (C) is obtained by enzymatic reaction with helicase. The band corresponding to the double-stranded nucleic acid in which the oligonucleotide having the sequence set forth in SEQ ID NO: 42 and the oligonucleotide having the sequence set forth in SEQ ID NO: 43 are base-paired is shown. In addition, the conceptual diagram of reaction in a present Example is shown in FIG. 6 (here (A), (B) and (C) represent the same nucleic acid as FIG. 5). From FIG. 5, it was found that the activity of helicase can be measured using nucleic acids having different sequences and lengths.

Claims (3)

A partially double-stranded nucleic acid (A) composed of a single-stranded nucleic acid (A-1) and a single-stranded nucleic acid (A-2), a single-stranded nucleic acid (B), and a helicase,
A method for measuring helicase activity, comprising measuring a double-stranded nucleic acid or a partially double-stranded nucleic acid (C) comprising a single-stranded nucleic acid (A-2) and a single-stranded nucleic acid (B). ,
The nucleic acid (A-1) comprises a sequence containing a sequence (helicase recognition sequence) necessary for an enzymatic reaction with helicase at the 5 ′ end and / or 3 ′ end,
The nucleic acid (A-2) comprises a sequence complementary to a portion other than the helicase recognition sequence in the nucleic acid (A-1),
The nucleic acid (B) comprises a sequence complementary to a part of the nucleic acid (A-2), or a sequence completely complementary to the nucleic acid (A-2), and (A-2 ) When nucleic acid base-paired with a nucleic acid of (), and does not contain a helicase recognition sequence,
The method as described above, wherein the nucleic acids (A-1), (A-2) and (B) are unlabeled nucleic acids. The measurement method according to claim 1, wherein the nucleic acid (C) is measured using electrophoresis. The method according to claim 1 or 2, wherein the helicase is a helicase derived from a virus of the Coronaviridae or Flaviviridae family, or a mutant, partial deletion, or fusion thereof. .

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