JPH1156398A - Assay of double-stranded nucleic acid splitting enzyme activity - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for the subject assay. SOLUTION: This method comprises the following three consecutive processes: (1) a single-stranded probe having, in the molecule, a recognition base sequence portion for a double-stranded nucleic acid splitting enzyme and a base sequence portion complementary to the above base sequence portion, and also having, on both ends, an energy donor and an energy acceptor, is prepared; (2) the recognition base sequence portion is hybridized, in the molecule, with the base sequence portion complementary thereto to form a hybrid; and (3) the change of the fluorescence spectrum based on the fluorescence resonance energy transfer(FRET) of the probe due to the photoexcitation of the energy donor by the decomposition of the hybrid due to nucleic acid splitting enzymatic reaction is assayed.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

[0001] The present invention relates to a method for measuring the activity of a nuclease, particularly a base sequence-specific double-stranded nuclease.

[0002]

2. Description of the Related Art In a method for measuring the activity of a nuclease, a fluorescence resonance energy transfer (fluoresc) between two fluorescent dyes is performed using a probe labeled with two fluorescent dyes.
A measurement method based on the phenomenon of energy resonance energy transfer (FRET) is known (SSGhosh,
PSEis, K. Blumeyer, K. Fearon, and DPMillar (19
94), Nucleic Acids Res., 22, 3155-3159). In this method, as shown in FIG. 1, an oligonucleotide probe to which a fluorescent energy donor dye (hereinafter, referred to as D) is bound, and an oligonucleotide probe to which a fluorescent energy acceptor dye (hereinafter, referred to as A) is bound. Is hybridized to a hybrid soybean body (double-stranded)
And a part of the nuclease to be measured (double-stranded nuclease) having a specific recognition base sequence. In such a hybridized substance, a FRET phenomenon occurs between D and A bound to each end, and a unique fluorescence spectrum is given. Further, after the specific site is cleaved by the nuclease, the above-mentioned FRET phenomenon is lost, and the above-mentioned specific fluorescence spectrum is lost.

[0003]

However, the method for measuring nucleolytic activity using the FRET phenomenon by a set of probes using two kinds of oligonucleotides as described above uses the two kinds of oligonucleotides in a measuring solution. It is necessary that the same number and the same number be present and that a hybridized body be formed. That is, when a protein that can bind to another single-stranded nucleic acid or the like is mixed in the measurement solution, or due to a change in hybridization conditions, one of the probes is present in the measurement solution as it is without hybridization. It is possible. In this case, there is a problem that the fluorescence from the non-hybridized probe becomes the background fluorescence for the measurement, and it becomes difficult to measure the nuclease activity using the FRET phenomenon.

[0004]

Means for Solving the Problems The present inventors have conducted intensive studies and have found a novel method for measuring the activity of nuclease which does not have the above-mentioned problems by using one kind of single-stranded oligonucleotide probe. The invention has been completed.

That is, the probe used in the method for measuring the activity of the nuclease according to the present invention is, as shown in FIG. 2, one kind of single-stranded oligonucleotide probe, and at least (1) the FRET phenomenon 1 for using
Having a set of D and A at the end of the oligonucleotide;
And (2) the probe has a specific base sequence that forms a base sequence portion as an enzyme reaction recognition portion of the nuclease to be measured when the probe forms a double-stranded structure in the molecule. Further, (3) the D and A are used when the probe forms a double-stranded structure in the molecule.
It is possible to take a spatial position that makes it possible.

When a probe having the above structure in which a hybrid is formed in a molecule is cleaved by the above-described specific enzyme reaction recognition portion by the nuclease reaction to be measured,
As shown in FIG. 2, the cleaved portion to which D or A is attached can no longer hybridize well and is released into solution. Therefore, the FRET phenomenon disappears.

[0007] As shown in FIG. 3, the fluorescence spectrum of the solution of the above nuclease reaction shows that fluorescence based on D before the enzyme treatment is hardly observed by FRET.
After the FRET phenomenon disappears by the enzyme treatment, fluorescence based on D is largely observed, and the reactivity of the nuclease can be measured.

That is, the present invention provides (1) a molecule comprising a double-stranded nuclease-recognizing base sequence portion in a molecule and a base sequence portion complementary to the base sequence portion, and having energy at both ends. By preparing a single-stranded probe having a donor and an energy acceptor, (2) in the molecule, the recognition base sequence portion;
The base sequence portion and the complementary base sequence portion are hybridized to form an intramolecular double-stranded structure. (3) Fluorescence based on fluorescence resonance energy transfer (FRET) of the probe caused by photoexcitation of the energy donor. It is intended to provide a method for measuring a double-stranded nuclease activity, which comprises measuring a change in spectrum of the intramolecular double-stranded structure due to the decomposition by the nuclease reaction.

[0009] The present invention also provides (1) a molecule comprising a recognition base sequence of the first double-stranded nuclease, a base sequence complementary to the base sequence, and a second double-stranded sequence. A single-stranded probe having a recognition base sequence portion of a strand nuclease and a base sequence portion complementary to the base sequence portion, and having, at both ends, an energy donor and an energy acceptor, (2)
In the molecule, the recognition base sequence part of the first double-stranded nuclease is hybridized with the complementary recognition base sequence part, and the recognition base sequence part of the second double-stranded nuclease is Forming an intramolecular double-stranded structure by hybridizing with a complementary base sequence portion; and (3) obtaining a fluorescence spectrum of the probe based on fluorescence resonance energy transfer (FRET) of the probe by photoexcitation of the energy donor. A method for measuring a double-stranded nuclease activity, comprising measuring a change due to degradation caused by the first nucleolytic enzyme reaction, the second nucleolytic enzyme reaction, or the third nucleolytic enzyme reaction. Is what you do.

[0010] The present invention further provides (1) a method comprising:
A double-stranded nuclease-recognizing base sequence portion, a base sequence portion complementary to the base sequence portion, a second double-stranded nucleolytic enzyme recognition base sequence portion, and a base sequence portion complementary to the base sequence portion. Base sequence, a recognition base sequence portion of the third double-stranded nuclease, and a base sequence portion complementary to the base sequence portion, and at both ends an energy donor and an energy acceptor. Preparing a single-stranded probe having: (2) hybridizing in a molecule with the recognition base sequence portion of the first double-stranded nuclease and the complementary recognition base sequence portion, The recognition base sequence of the third double-stranded nuclease is allowed to soy with the complementary base sequence of the double-stranded nuclease and the complementary base sequence and the complementary base of the third base. Hybridizing with the sequence portion to form an intramolecular double-stranded structure; Fluorescence spectrum based on fluorescence resonance energy transfer (FRET) of the probe by photoexcitation of Azukakarada, the said probe first nuclease reaction or the second nuclease reaction or the third
A method for measuring the activity of a double-stranded nuclease, characterized by measuring a change caused by the degradation of the nuclease by a nucleolytic enzyme reaction.

Further, in any one of the above methods, (1) the recognition base sequence portion of the double-stranded nuclease may be:
Another object of the present invention is to provide a method characterized by having a base sequence for recognition of the double-stranded nuclease and two to five bases on both sides of the recognition sequence.

Further, the present invention provides a method wherein the energy donor comprises a fluorescein-based fluorophore and the energy acceptor comprises a rhodamine-based fluorophore.

Hereinafter, the present invention will be described in more detail.

(Fluorescence Resonance Energy Transfer, FRET) The FRET phenomenon in the present invention is a phenomenon relating to fluorescence (Stryer, L., Ann. Rev. Biochem, 47, 1978, 819-846;
Fluorescence Microscopy of Living Cellsin Culture-
Part B. Taylor, DL and Wang, Y. eds., Academic Pre
ss, New York, 220-245, 1989). The fluorescence spectrum of one fluorophore (hereinafter, referred to as an energy donor, an energy donor dye, an energy donor, a donor, D) is different from that of another fluorophore (an energy acceptor). Body, energy acceptor dye, energy acceptor, acceptor, A) and the donor and acceptor are close to each other, the excitation of the donor induces fluorescence from the acceptor. This means that the fluorescence intensity from the donor itself decreases.

[0015] FRET is known to be very sensitive to the distance between the donor and acceptor, generally proportional to the 10 ^-6 square of the distance between them.

The FRET phenomenon is caused by various organisms,
It is used in cell biology research (for example, structural study of Hammerhead ribozyme in solution, concentration study of cAMP in vivo, membrane fusion, retroviral protease, nucleic acid structure and nucleotide sequence, and This is the same phenomenon as that of intracellular oligonucleotide hybridization.

(Energy Donor (Donner), D; Energy Acceptor (Acceptor), A) As the energy donor (donor) and energy acceptor (acceptor) usable in the present invention, FRET can be used. The combination is not particularly limited.

Dyes having various fluorophores can be used. For example, fluorescein dyes and rhodamine dyes can be suitably used.

In the present invention, it is particularly preferable to use a dye having a fluorescein fluorophore as a donor and a dye having a rhodamine X fluorophore as an acceptor.

The absorption spectra of fluorescein and rhodamine X have absorption peaks at 497 and 586 nm.

On the other hand, fluorescein shows a fluorescence peak at 523 nm when excited at 494 nm. Rhodamine X shows a fluorescence peak at 610 nm when excited at 585 nm.

If the two fluorophores are at a distance sufficient to cause FRET, the fluorescence intensity based on fluorescein decreases with the transfer of the fluorescent energy from fluorescein as a donor to rhodamine X as an acceptor. However, the fluorescence intensity based on rhodamine X will increase.

(Probe) A single-stranded probe that can be used in the method of the present invention has D and A bonded to at least both ends to use the FRET phenomenon, as schematically shown in FIG. And,
It has a base sequence for recognizing double-stranded nuclease and a recognition base sequence complementary thereto in the molecule.
Such a probe, under normal hybridization conditions, the recognition base sequence and its complementary base sequence hybridize in the molecule to form an intramolecular double-stranded structure, and the molecule has a structure similar to a hairpin structure. (FIG. 2). Therefore, D and A bound to both ends of the probe are close enough in spatial position to be a position sufficient to cause the FRET phenomenon. In this case, when light is excited with respect to D, the excitation energy resonates and moves to A to give a characteristic fluorescence spectrum.

FIG. 3 schematically shows a change in the fluorescence spectrum based on the FRET. When the FRET phenomenon occurs, fluorescence from D based on photoexcitation of D is hardly observed. On the other hand, when the FRET phenomenon disappears, strong fluorescence from D occurs based on the photoexcitation of D, which is observed as a large change in the fluorescence spectrum. The change in the fluorescence spectrum makes it possible to confirm the presence of the test nuclease.

That is, as shown in FIG. 2, the probe of the present invention is reacted with a double-stranded nuclease to cut (decompose) at the recognition sequence portion in the probe.
Occurs. After this degradation occurs, the oligonucleotide to which D and A are bound no longer has enough base pairs to retain the hybridized form and is released into solution. As a result, the above-mentioned FRET phenomenon disappears, and the fluorescence spectrum is greatly changed (FIG. 3).

Further, the probe according to the present invention can be intramolecularly hybridized under a condition of sufficiently low concentration in a solution and under an appropriate temperature condition, so that the hairpin hybridized intramolecularly at the recognition sequence portion can be obtained. It is possible to obtain only those having a structure. In a preferred embodiment, the recognition sequence portion has not only the recognition base sequence of the double-stranded nuclease but also some further base pairs at both ends thereof. That is, it is a structure in which the enzyme further has some extra base pairs necessary for performing the enzyme reaction. Although the number depends on the test nuclease, it is usually 2 to 5
Preferably there are two base pairs.

Further, the structure (spacer portion) between D or A of the probe according to the present invention and the recognition sequence portion is not particularly limited, and various chemical structures are possible. When it has, it is preferably a base pair for the purpose of forming a base pair and stabilizing the probe. In the case of base pairs, if the number of base pairs is too large, a hybridized body exists sufficiently stably even after the probe is decomposed at the enzyme reaction recognition part by the enzyme reaction, and FRET of No disappearance is observed, which is not preferable. Therefore, the number of bases is preferably selected to such an extent that a double strand cannot be retained depending on the base pairs remaining in each fragment when degraded by a nuclease. This number varies depending on the temperature and the type of base (GC content), but can be easily selected by a generally known method (PCR: Clinical Diagnosti).
cs and Research, A. Rolfs, I. Schuller, U. Finckh,
I. WeberRolfs eds., P.11 Sringer Laboratory (199
2)). Specifically, under normal temperature conditions (25-40 ° C), 1
It is preferably 5 bases or less. The probe according to the present invention has a hairpin structure after forming a double-stranded structure in the molecule. For this purpose, it is sufficient if the length of the molecule between the recognition sequence portion and its complementary base sequence portion is too short. It is not preferable because a proper hairpin structure cannot be obtained. Therefore, in order to maintain a stable hairpin structure, it is necessary that the recognition sequence is adjacent to the recognition sequence at least one length, preferably at least three lengths in terms of nucleobases.

(Single-strand probe is another example) As described above, the structure of the probe according to the present invention does not need to have the enzyme reaction recognition portion at one place, and a plurality of enzyme reactions can be included in the same molecule. It is possible to provide a recognition unit. Specifically, as schematically shown in FIGS. 4A and 4B for another example of the probe of the present invention, a probe having two or three enzyme reaction recognition units. Can be adjusted. The use of such a probe makes it possible to measure the activities of a plurality of types of double-stranded nucleases. That is, since the probe has a plurality of double-stranded nuclease-recognizing enzyme recognition base sequence portions, it becomes a probe having a plurality of hairpin structures by intramolecular hybridization (FIG. 4).
(A), (B)). In this case, when the probe is degraded by the nuclease, the phenomenon based on FRET disappears.

FIG. 5 schematically shows a probe capable of simultaneously detecting the activity of any of the three types of double-stranded nucleases and the action thereof. FRET disappears when each of the enzymes 1, 2, and 3 is decomposed and cleaved by a probe having a recognition base sequence portion of three types (1, 2, and 3, respectively) of double-stranded nucleases. Indicates that

(Method for Preparing Single-Stranded Probe) In the present invention, D and A are used as oligonucleotides 5 ′ or 3 ′.
The method of binding to the terminal is not particularly limited, and can be synthesized by a general organic synthesis method or an enzymatic reaction using an appropriate derivative.

In the present invention, a nucleotide having a rhodamine X fluorophore or a rhodamine fluorophore can be bound to an oligonucleotide having a fluorescein fluorophore at the 5 ′ end by a chemical synthesis method or by an enzymatic reaction. is there.

Similarly, in the present invention, an oligonucleotide having a rhodamine X fluorophore at the 3 ′ end (9-m
er), a nucleotide having a fluorescein fluorophore can be synthesized by chemical synthesis or enzymatic chemistry.

In the present invention, the nucleotide sequence of the oligonucleotide can be selected according to the purpose of use, and is not particularly limited.

(Measurement of FRET phenomenon, measurement of fluorescence spectrum)
There is no particular limitation on the method of measuring and comparing the fluorescence spectra from the different fluorophores and their changes.

For example, the ratio of the respective fluorescence intensities from two kinds of fluorophores under the condition that FRET is possible, and F
A method of comparing the ratio of the fluorescence intensities from two kinds of fluorophores under the condition where RET does not occur can be suitably used.

In this case, in order to perform the measurement with higher sensitivity, it is possible to suitably use a filter for selecting an excitation wavelength, a selective measurement of fluorescence, and a means for simultaneously measuring two types of fluorescence. The fluorescence spectrum measuring device used in the method according to the present invention is not particularly limited, and an ordinary device can be preferably used.

(Some application examples of the method according to the present invention)
According to the present invention, two types of fluorescent dyes which generate FRET are bound to both ends, and a nucleolytic enzyme activity measuring probe having a hairpin structure by intramolecular hybridization in a solution is used as a substrate, and a specific spectroscopic method is used. The method for measuring nuclease activity is applicable, for example, in the following cases.

(1) Since the types of restriction enzymes to be retained for microorganisms such as Escherichia coli and viruses are determined according to their types (for example, Table 1), harmful microorganisms and pathogenic bacteria can be a simple means as a screening method for identifying the types. .

(2) Since the fluorescence intensity changes greatly only by mixing one type of substrate, the method can be applied to a simple, short-time and highly sensitive method for measuring nuclease activity.

(3) Since the degrading enzyme activity can be measured even in the presence of other biological components such as proteins and nucleic acids, the separation and purification steps of the degrading enzyme become unnecessary, and the sample to be inspected for fluorescence measurement is not required. Preprocessing is simplified. In addition, by introducing a probe into a cell, the enzymatic activity in a living biological sample can be measured.

[0041]

That is, the probe used in the method for measuring the activity of the nuclease according to the present invention comprises, as shown in FIG.
Kinds of single-stranded oligonucleotide probes, comprising at least (1) a set of D
And A at the end of the oligonucleotide; and
(2) when the probe forms a double-stranded structure in the molecule,
It has a specific base sequence that forms a base sequence portion as an enzyme reaction recognition portion of the nuclease to be measured. Further, (3) such D and A can take a spatial position such that FRET from D to A becomes possible when the probe forms a double-stranded structure in the molecule.

When the probe having the above structure in which a hybrid is formed in the molecule is cleaved by the above-mentioned specific enzyme reaction recognition portion by the nuclease reaction to be measured,
As shown in FIG. 2, the cleaved portion to which D or A is attached can no longer hybridize well and is released into solution. Therefore, the FRET phenomenon disappears.

The 5 'and 3' ends of the oligonucleotide are labeled with two different fluorescent dyes (energy donor and energy acceptor), and the fluorescence resonance energy in the molecule by these two fluorophores is determined. Move (FRE)
By comparing the fluorescence characteristics based on T), the degradation activity of the oligonucleotide can be measured.

[0044]

DESCRIPTION OF THE PREFERRED EMBODIMENTS The present invention will be described below in detail with reference to embodiments. However, the present invention is not limited to this embodiment.

(Example 1) Preparation of PvuII probe and HindIII probe The following nucleotide sequence was prepared by a chemical synthesis method, and fluorescein as an energy donor was bound to the 3 'end and 5'
Oligonucleotides having an amino group bonded to the terminal were synthesized. By reacting rhodamine X isothiocyanate with this amino group, the 5 ′ of the oligonucleotide is
Rhodamine X was bound to the end. The obtained probe was purified by high performance liquid chromatography.

PvuII probe : Fluorescein as an energy donor (dye) at the 5 ′ end and an energy acceptor (dye) at the 3 ′ end The following 33mer bound with Rhodamine X as)).

5'-TTTT CAGCTG GGGGTAGATCCCC CAGCTG AAAA-
3 ′ (the underlined portion indicates the recognition base sequence of the restriction enzyme PvuII).

HindIII probe : Fluorescein as an energy donor (dye) having a recognition base sequence AAGCTT of a restriction enzyme HindIII in a part thereof and an energy acceptor (dye) at a 3 ′ end thereof. The following 33mer bound with Rhodamine X as)).

5'-TTTT AAGCTT GGGGTAGATCCCC AAGCTT AAAA-
3 ′ (the underlined portion indicates the recognition base sequence of the restriction enzyme HindIII).

For the fluorescence spectrum, a Hitachi spectrophotometer 850 was used.

Example 2 Reaction of PvuII Probe with Various Restriction Enzymes The effect of the restriction enzyme PvuII on the fluorescence spectrum of the PvuII probe was examined. 20nM PvuII probe and 2 units of Pvu
II (purchased from Takara Shuzo Co., Ltd.) with 10 mM TrisHCl (pH 7.5)
The reaction was carried out at 25 ° C. in a reaction solution composed of components of 10 mM MgCl ₂ , 1 mM DTT, and 50 mM NaCl (the unit of the enzyme, that is, the activity was according to Takara Shuzo Co., Ltd. product data). Two hours later, the fluorescence spectrum when excited by light having a wavelength of 492 nm was measured using a fluorimeter, and the results are shown in FIG. As a control experiment, the same treatment was performed using a solution containing no enzyme. In the fluorescence spectrum from the control solution, fluorescence having a peak wavelength of 518 nm due to the donor was hardly observed, and it was recognized that FRET occurred (indicated as control in FIG. 6).

On the other hand, in the solution containing PvuII, the fluorescence of the donor was significantly increased (shown as PvuII in FIG. 6). This means that FRET was eliminated (disappeared) due to the presence of PvuII, and indicates that the enzyme decomposed (cleaved) the PvuII probe.

It was confirmed by using various restriction enzymes that the above change was specific to the recognition sequence of the restriction enzyme. The restriction enzymes used are shown in Table 1 below (purchased from Takara Shuzo Co., Ltd.).

[0054] Since the recognition sequences of HindIII and SacI differ in the nucleotide sequences at both ends of the recognition sequence inserted into the PvuII probe, these two types of restriction enzymes cannot cleave the PvuII probe. However, the recognition sequence of AluI is the internal 4 base AGC of the recognition sequence of PvuII.
Since it is the same as T, the PvuII probe can be cleaved.

After 2 units of each enzyme were reacted with a 20 nM PvuII probe and allowed to stand at 25 ° C. for 2 hours, the fluorescence spectrum was measured. The results are shown in FIG. The reaction solution used was 10 mM TrisHCl (pH 7.5), 10 mM for SacI and AluI.
Consists of MgCl ₂ , 1 mM DTT, 10 for HindIII and PvuII
mM TrisHCl (pH 7.5), 10 mM MgCl ₂ , 1 mM DTT, 50 mM NaCl
Consists of As a result of the experiment, the addition of HindIII and SacI showed the same fluorescence spectrum as the experimental result of the control without addition of the enzyme, and did not change the fluorescence intensity of the donor dye (FIG. 6, HindIII and SacI). ), The addition of AluI, like PvuII, caused a significant increase in the emission intensity of the donor (indicated by AluI in FIG. 6). From these fluorescence spectra, the wavelengths 518 nm and 609
The ratio of the fluorescence intensity at nm (the fluorescence peak wavelength of the receptor dye) was calculated, and is shown in FIG. 7 as the ratio of the fluorescence intensity for each enzyme. Here, PvuII and control without enzyme, low values of HindIII and SacI
It is clear that the ratio of AluI is significantly higher. Therefore, Pv
This indicates that cleavage (decomposition) of the uII probe is specific to the recognition sequence of a restriction enzyme (double-stranded nuclease).

(Example 3) Reaction between HindIII probe and various restriction enzymes Restriction enzyme-recognition sequence-specific cleavage is not limited to a probe for PvuII, but can be a probe having a recognition sequence for another restriction enzyme. PvuI to find out
Using a HindIII probe in which the recognition sequence of the I probe was changed to a base sequence recognized by HindIII, a fluorescence measurement experiment of an enzyme reaction was performed. The base sequence of the HindIII probe was 5'-TTTT AAGCTT GGGGTAGATCCCC AAGCTT AAAA-3 'as shown above.
(The underlined portion is the restriction enzyme HindIII recognition sequence). 2 units of each restriction enzyme shown in Table 1
After reacting with 20 nM HindIII probe and reacting in each of the solutions shown in Example 2 for 2 hours, excitation was performed with light at 492 nm and the fluorescence spectrum was measured. The results are shown in FIG. The ratio of the fluorescence intensities at 518 nm and 609 nm was determined from the obtained spectrum and is shown in FIG. HindIII probe is HindI
Although the fluorescence intensity ratio was increased by the addition of II and AluI, PvuII and Slu
The addition of acI showed the same low value as the control to which no restriction enzyme was added. This result indicates that AluI recognized and cleaved the middle sequence of the HindIII recognition sequence, but PvuII and Sac
The I recognition sequence indicates that the HindIII probe could not be cleaved.

Example 4 A method for measuring nuclease activity using a probe according to the present invention labeled at both ends with a FRET donor (dye) and an acceptor (dye) is not limited to restriction enzymes, The double-stranded DNA degrading enzyme BAL31n is also applicable to double-stranded nucleic acid enzymes that are not sequence-specific.
It was confirmed using uclease (hereinafter referred to as BAL31).

Each of the PvuII probe and the HindIII probe was dissolved in a buffer containing 2 units of BAL31 to a final concentration of 20 nM. Buffer components are 20 mM TrisHCl (pH 8.0), 1
2 mM CaCl ₂ , 12 mM MgCl ₂ , 0.6 M NaCl, 1 mM EDTA.
After leaving at room temperature (25 ° C.) for 2 hours, excitation was performed at a wavelength of 490 nm, and the fluorescence spectrum was measured. The results are shown in FIG.

In both probes, a change in the fluorescence spectrum was clearly observed by the BAL31 treatment. Wavelength 51
The ratio of the fluorescence intensity at 8 nm and 609 nm increased from 0.69 before the enzyme treatment to 7.73 and 11.2 times after the treatment in the PvuII probe, and increased to 6.88 and 12.1 times from 0.57 before the treatment to 0.58 in the HindIII probe after the treatment. did. This result shows that the method for measuring the activity of a probe according to the present invention by FRET measurement is not limited to restriction enzymes, and can be effectively applied to double-stranded nucleases that do not depend on the base sequence. .

[0060]

[Sequence list]

SEQ ID NO: 1 Sequence length: 33 Sequence type: Nucleic acid Number of strands: Double strand Topology: Linear Sequence type: DNA Sequence characteristics: Fluorescein binds to 5 'end, 3'
Rhodamine X binds to the end Sequence TTTTCAGCTG GGGGTAGATC CCCCAGCTGA AAA 33 SEQ ID NO: 2 Sequence length: 33 Sequence type: Nucleic acid Number of strands: Double strand Topology: Linear Sequence type: DNA Sequence characteristics: 5 ' Fluorescein binds to the end, 3 '
Rhodamine X binds to the end Sequence TTTTAAGCTT GGGGTAGATC CCCAAGCTTA AAA 33

[Brief description of the drawings]

FIG. 1 is a diagram schematically showing a conventional example of a probe composed of a pair of two oligonucleotides utilizing the FRET phenomenon.

FIG. 2 shows that the probe according to the present invention (FIG. 2 (A)) hybridizes in a molecule to form a hairpin structure, and thus the probe has an FR.
FIG. 2 is a diagram schematically showing that the ET phenomenon occurs (FIG. 2 (B)), the nuclease recognition sequence is cleaved by the double-stranded nuclease, and the FRET phenomenon disappears (FIG. 2 (C)). .

FIG. 3 is a diagram showing that the fluorescence spectrum of the probe according to the present invention changes based on disappearance of the FRET phenomenon due to treatment with a nuclease.

FIG. 4 shows an example of a probe according to the present invention, in which (A) is a probe having two types of nuclease recognition sequences (shown as 1, 2 recognition sequences, respectively); (B) shows three types of nuclease recognition sequence parts (shown as 1, 2, and 3 recognition sequences, respectively)
It is a figure which shows typically the probe which has.

FIG. 5 is a diagram schematically showing cleavage by a restriction enzyme of a probe according to the present invention having three types of nuclease recognition sequence portions (represented as recognition sequences for restriction enzymes 1, 2, and 3, respectively). It is.

FIG. 6 is a diagram showing a change in fluorescence spectrum of a PvuII probe treated with a restriction enzyme (PvuII, HindIII, SacI, AluI).

FIG. 7 is a diagram showing the ratio of the fluorescence intensity of a PvuII probe treated with a restriction enzyme (PvuII, HindIII, SacI, AluI) at wavelengths of 518 nm and 609 nm.

FIG. 8 is a diagram showing a change in fluorescence spectrum of a HindIII probe treated with a restriction enzyme (PvuII, HindIII, SacI, AluI).

FIG. 9 is a diagram showing the ratio of the fluorescence intensity at 518 nm and 609 nm of a HindIII probe treated with restriction enzymes (PvuII, HindIII, SacI, AluI).

FIG. 10 shows a probe (P
FIG. 4 is a diagram showing changes in the fluorescence spectrum of a vuII probe and a HindIII probe).

Claims (5)

[Claims] 1. (1) a molecule having a base sequence-specific double-stranded nuclease-recognizing enzyme in a molecule, and a base sequence portion complementary to the base sequence portion, and A single-stranded probe having an energy donor and an energy acceptor was prepared, and (2) in the molecule, the recognition base sequence portion was hybridized with a base sequence portion complementary to the base sequence portion. (3) a fluorescence spectrum based on fluorescence resonance energy transfer (FRET) of the probe caused by photoexcitation of the energy donor by the nucleolytic enzyme reaction of the intramolecular double-stranded structure; A method for measuring a double-stranded nuclease activity, which comprises measuring a change due to degradation. 2. The method according to claim 1, wherein: (1) a base sequence-specific first
A double-stranded nuclease-recognizing base sequence portion, a base sequence portion complementary to the base sequence portion, a second double-stranded nuclease-recognizing base sequence portion, and a base sequence portion complementary to the base sequence portion. And a single-stranded probe having an energy donor and an energy acceptor at both ends.
(2) In the molecule, the recognition base sequence of the first double-stranded nuclease is hybridized with the complementary recognition base-sequence and the recognition base sequence of the second double-stranded nuclease. And the complementary base sequence portion to form an intramolecular double-stranded structure, and (3) a fluorescence spectrum based on fluorescence resonance energy transfer (FRET) of the probe caused by photoexcitation of the energy donor. A method for measuring a double-stranded nuclease activity, which comprises measuring a change in the probe due to decomposition by the first nucleolytic enzyme reaction or the second nucleolytic enzyme reaction. 3. The method according to claim 1, wherein (1) a base sequence-specific first
A double-stranded nuclease-recognizing base sequence portion, a base sequence portion complementary to the base sequence portion, a second double-stranded nuclease-recognizing base sequence portion, and a base sequence portion complementary to the base sequence portion. Base sequence, a recognition base sequence portion of the third double-stranded nuclease, and a base sequence portion complementary to the base sequence portion, and at both ends an energy donor and an energy acceptor. Preparing a single-stranded probe having: (2) hybridizing in a molecule with the recognition base sequence portion of the first double-stranded nuclease and the complementary recognition base sequence portion, The recognition base sequence of the double-stranded nuclease is hybridized with the complementary base sequence, and the recognition base sequence of the third double-stranded nuclease is hybridized with the complementary base sequence. To form an intramolecular double-stranded structure, (3)
The first nucleolytic enzyme reaction, the second nucleolytic enzyme reaction, or the third nucleolytic reaction of the probe in a fluorescence spectrum based on fluorescence resonance energy transfer (FRET) of the probe due to photoexcitation of the energy donor A method for measuring a double-stranded nuclease activity, which comprises measuring a change due to decomposition by an enzyme reaction. 4. The recognition base sequence of the double-stranded nuclease has a recognition base sequence of the double-stranded nuclease and 2 to 5 bases on both sides of the recognition sequence. The method according to any one of claims 1 to 3, characterized in that: 5. The method according to claim 1, wherein said energy donor comprises a fluorescein-based fluorophore and said energy acceptor comprises a rhodamine-based fluorophore.