WO1996025518A1

WO1996025518A1 - Probe for use in nucleic acid analysis and detecting method

Info

Publication number: WO1996025518A1
Application number: PCT/JP1996/000297
Authority: WO
Inventors: Masayuki Masuko; Katsuyoshi Ebata
Original assignee: The Society For Techno-Innovation Of Agriculture, Foresty And Fisheries
Priority date: 1995-02-17
Filing date: 1996-02-13
Publication date: 1996-08-22
Also published as: AU694313B2; AU4633896A

Abstract

A probe for detecting nonradioactive nucleic acids by the hybridization technique, and a highly sensitive and highly selective method for the detection. The probe comprises a set of two or more oligonucleotide probes that can hybridize perfectly complementarily with the specific portion of the sequence of the consecutive bases of the target nucleic acid, each oligonucleotide probe being labeled at the 5' or 3' end thereof with a chromophoric group having a suitable spatial arrangement so that adjacent chromophoric groups can yield an excimer or the like when each probe hybridizes with the target nucleic acid. More specifically, although the probe of the invention comprises a set of two or more labeled probes and the labeling groups to be detected are present each on a different probe, the labeling groups take such a spatial arrangement that the two chromophoric groups present on the two probes which have come to exist adjacent to each other only after the hybridization with the target nucleic acid induce unique phenomena such as excimer fluorescence, thereby enabling the target nucleic acid to be detected with a high recognizability. Thus the probe of the invention enables the erroneous recognition which has been problematic heretofore to be reduced remarkably and the types of utilizable labeling groups to be varied widely. Further it is possible to improve the detection sensitivity remarkably because it is possible to reduce the background noise remarkably. In addition, it is possible to discriminate a number of (single-base variation) nucleic acids that are different from one another in only one base present at a specific position to be detected, which has been difficult heretofore.

Description

Description Probe for nucleic acid analysis and detection method

An object of the present invention is to provide a probe for nucleic acid analysis having high sensitivity and high selective nucleotide sequence recognition without using radioisotopes, and a detection method using the same. Background art

In genetic engineering, the technique called nucleic acid hybridization, which is used to detect specific genes and nucleic acids from cells and viruses, depends on the purpose, target gene, and detection method. Many methods have been developed and used.

In particular, various methods have been developed for identifying and further quantifying the hybridized target substance. Generally, in this detection method, it is necessary to use a polynucleotide that hybridizes with the nucleic acid to be detected as a nucleic acid detection probe and to label a part thereof. For example, it is one of the most sensitive detection methods, and a commonly used method is to use a radioisotope. Non-radioactive labeling methods include labeling of fluorescent substances, labeling of enzymes, and the like, and methods of observing phenomena based on the interaction between label groups.

In the above-mentioned conventional hybridization method, a complementary polynucleotide is used as a probe for detecting one kind of nucleic acid with respect to a target nucleic acid (target or target polynucleotide). The probe must be used in excess of the target to perform hybridization, and the probe polynucleotide for nucleic acid detection needs to be removed by washing or the like before detection, resulting in complicated operation. And lack of agility. As a recent trend, it does not use radioisotopes, but has practical sensitivity (detection limit) performance and high recognition (recognition of a single base difference) performance, and after hybridization as described above. It has been particularly desired to develop a detection method that utilizes a phenomenon that can be directly measured without the need to wash and remove excess probe.

In order to partially attain this object, several methods are already known, which enable detection without the need for an operation for removing excess probe. USP5,332,659 is an example of the use of the phenomenon based on the excimer formation of two chromophores. In this method, one kind of probe for detecting a polynucleotide, which is preliminarily provided with two or more fluorescent labels in a single strand, is used, and the change in excimer light intensity that occurs when hybridization with the target nucleic acid is performed. A method for detecting the target nucleic acid by observation is disclosed.

Furthermore, when a long target nucleic acid is detected by hybridization using an oligonucleotide as a probe, the purpose of the present invention is to reduce the number of misrecognitions or to provide recognition with a single base difference (point mutation recognition). It is apparent that it is more preferable to use one set of two or more kinds of probes for one target than one kind of probe. Disclosure of the invention

Based on this viewpoint, by measuring the phenomenon that occurs when a plurality of, for example, two polynucleotide detection probes are hybridized with a target nucleic acid, each label group provided on the target is determined by measuring the phenomenon. Methods for detecting nucleic acids are known. A pair of energy donors (D) / Axceptors (A) are used as label groups, each of which is attached to a separate oligonucleotide. It is also known that the same energy transfer is used, but one of them is enzymatically labeled. That is, EP0229943A2 discloses a labeling method based on D / A. However, the spectrum change due to the energy transfer between D / A is essentially not so sensitive to the position between D and A, and the fluorescence spectra of donor and executor overlap so much that the difference of one base Accurate recognition is extremely difficult.In addition, EP0070685B1 is unsuitable for hybridizations that often require thermal treatment because they are labeled with an enzyme that is not thermally stable in nature. You. In this method, the energy donor is not in the probe but is a luminescent substance such as luminol mixed in the measurement solution as a substrate for the enzyme. These are not sensitive enough to identify the position between the enzyme and xuebu because they diffuse in solution, and are unsuitable for the hybridization method that requires accurate recognition of a single base difference. . In view of the above problems of the conventional method, the present invention provides a non-radioactive, hybridization-based probe for nucleic acid detection, which is highly sensitive and can recognize a difference of one base. It is. Furthermore, after the hybridization operation, the complex that has hybridized with the probe can be specifically detected as it is without washing away the excessively coexisting probe. That is, the configuration of the probe according to the present invention is an oligonucleotide probe consisting of one or more sets of two or more oligonucleotides that hybridize completely complementary to a continuous specific base sequence portion of the target nucleic acid. Labeled at the 5 'or 3' end with a chromophore group that has an appropriate spatial arrangement so that when each probe hybridizes with the target nucleic acid, an excimer can be formed. .

The structure of the above-mentioned probe according to the present invention is not at all expected from a conventionally known method as described below, and further, is not expected at all in the effect based on the structure. That is, for the purpose of recognizing even a single base difference, a set of two or more labeled probes is used, and the phenomenon of the label groups to be detected is the Extremely effective despite the presence of As a result, excimer fluorescence is induced, and the fluorescence is easily observed. In other words, originally, a single chromophore on another probe adopts a spatial arrangement such that it efficiently forms the excimer-fluorescence with the chromophore on the adjacent probe for the first time by hybridizing with the target nucleic acid. Based on this, it is completely unpredictable that the target nucleic acid will be detectable with extremely high recognizability.

Hereinafter, the configuration of the present invention and the effects of the present invention based thereon will be described.

(1) False recognition problem

In the detection of a target nucleic acid based on a conventionally known hybridization method, when the number of the target nucleic acid is one, and particularly when the target nucleic acid is long, a pseudo-nucleotide having a certain degree of complementarity with the target nucleic acid is used. The nucleic acid can be hybridized (False Hybridization in FIG. 1), resulting in false recognition. One way to avoid this general problem is to label each probe as a set of two or more probes instead of single-stranded (Figure 2), so that both are correctly targeted nucleic acids. It is to utilize the fact that a specific phenomenon occurs between two labeling groups only in the case of hybridization (True Hybridization in Figure 2). That is, in the case of false recognition, this specific phenomenon does not occur (False Hybridization in Fig. 2). This method is not necessarily limited to two probes, and it is possible to use more probes simultaneously if necessary (Fig. 3). In this case, the recognition is correctly performed only when all the specific phenomena generated between the respective labeling groups can be simultaneously observed, and it is possible to significantly reduce false recognition.

(2) Selectability of labeling group

In addition, the available labeling groups can be selected as broadly as possible. Is desirable. Further, at the same time, those that prevent the labeling group from hybridizing with the target nucleic acid cannot be used, and the labeling group is not hybridized with the nucleic acid base-pairing side based on hydrophobic interaction or the like at the time of hybridization. You must also avoid car navigation. Conventionally known embodiments in which two or more labeling groups are provided in the middle of a single-stranded probe make it considerably difficult to create a molecular design that satisfies these conditions. On the contrary, in the probe according to the present invention comprising a separate set of a plurality of probes, the restriction can be relaxed. In other words, since only one additional labeling group needs to be provided at the end of each probe, a variety of binding modes known in the art can be used, and a wide range of specific phenomena to be measured can be selected. It is also possible to design an optimum chemical structure in advance for measuring the phenomenon. For example, the possibility of selecting the chemical structure of the linker, the possibility of optimizing the length of the linker, the chemical stability of the labeling group, the temperature stability, the storage stability, etc. can be freely selected.

(3) Detection sensitivity

In addition, as a general requirement of the hybridization method, it is necessary to reduce background noise as much as possible in order to increase detection sensitivity. When one type of probe with a label is used, the unhybridized probe itself inherently causes a phenomenon to be measured, and the degree of the change varies depending on the hybridization. Is the basis of detection. Therefore, in order to reduce the above-mentioned noise, it is desirable that a specific phenomenon can be observed only when the target nucleic acid is correctly hybridized, and specific fluorescence based on simultaneous hybridization of one set of plural probes is required. The method of emitting is preferable in this respect.

Further, in the method based on one set of multiple probes, hybridization is performed for each probe, and impurities and impurities are not hybridized. 9 It is also possible to remove one target nucleic acid, mixed probe, etc. by washing treatment or the like, and further reduce the background to the minimum.

(4) Recognition of single base difference

Further, when it is desired to distinguish nucleic acids that differ only in one base at a specific position to be detected (single base mutation), it is difficult to selectively detect with a single-stranded probe. On the other hand, a method in which one probe and two probes are hybridized so as to sandwich the mutated nucleobase, and a specific phenomenon occurs only when adjacent terminal label groups come close to each other, is a method in which a single base is used. The difference can be detected. Excimer-fluorescence, which is known to determine the formation of a specific phenomenon on the order of several angstroms, is suitable as this specific phenomenon. By using the excimer-fluorescence phenomenon, the spatial The difference (several angstroms) can be recognized sharply. On the other hand, when using the energy transfer phenomenon (A / D) or when using enzymatic reactions and chemical luminescence, it is possible to recognize the difference The range is far greater than the excimer described above, ranging from tens to hundreds of angstroms. Therefore, in one of the objects of the present invention, in a detection method capable of recognizing a single base difference, it is most preferable to use excimer formation. From the above, a method using a pair of two probes, which are provided with a labeling group and have a configuration capable of causing a specific phenomenon only when correctly hybridizing, has been conventionally used. It is clear that some problems of the law can be greatly improved. BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a diagram showing hybridization when a single-stranded nucleic acid detection probe is used, in which a true target nucleic acid and a false target nucleic acid are each hybridized. It is a figure showing a situation of erroneous recognition which may occur at the time of zoning.

FIG. 2 is a diagram showing hybridization when a set of two types of nucleic acid detection probes according to the present invention are used.Even if one of the false target nucleic acids is hybridized, erroneous recognition may occur. FIG.

FIG. 3 shows an embodiment in which an extremely long target nucleic acid is detected using one or more sets of three nucleic acid detection probes in order to reduce the possibility of misrecognition. FIG. 4 is a diagram showing changes in the fluorescence spectrum of mixed solutions of various concentrations of the target oligonucleotide and two types of nucleic acid detection probes.

FIG. 5 is a diagram showing a change in relative excimer fluorescence intensity (at 495 nm) in a fluorescence spectrum of a mixed solution of a target oligonucleotide having a seed concentration and two kinds of nucleic acid detection probes.

FIG. 6 is a diagram showing the effect of the length of the linker. Effect of pyrenealkyl iodoacet amide- introduced 16-mer probe on excimer formation. Here, PIA, N- (l-pyrene) iodoacetamide; PMIA, N- (l-pyrenemethyl) iodoacetamide; PEIA, N- (1-pyreneethyl) iodoacetamide; PPIA, N- (1-pyrenepropyl) iodoacetamide are shown.

FIG. 7 shows that it is acceptable to detect a target or a so-to-nucleic acid having a base that does not hybridize between two probes due to point mutation using the probe according to the present invention. It is. Pyrenemethyl iodoacetamide-introduced 16-mer and pyrene butyric acid hydrazide-introduced 16-mer were used. The 32-mer evening sequence is continuous.

FIG. 8 shows that excimer fluorescence is significantly reduced when there are bases that do not form base pairs between the two probes, indicating that the present invention can be used to detect point-mutated nucleic acids. It shows.

FIG. 9 is a view showing the structure of (Compound 4).

FIG. 1 is a diagram showing the structure of (Compound 5). FIG. 11 is a diagram showing the structure of (Compound 6). BEST MODE FOR CARRYING OUT THE INVENTION

The nucleic acid analysis probe according to the present invention is a nucleic acid analysis probe 1 and a nucleic acid analysis probe 2 for detecting a polynucleotide having q base sequences, the first nucleic acid analysis probe The base sequence of 1 is a complementary base sequence to r base sequences (r is an integer of 1 or more and (q-1) or less) continuous from the 5 'end of the polynucleotide, and the probe 1 Has a coloring group molecule from the 5 ′ end of the polynucleotide via a chain-substituting group, and the base sequence of the second nucleic acid analysis probe 2 is (r) from the 5 ′ end of the polynucleotide having q base sequences. + l) a base sequence complementary to the base sequence between the q-th and q-th bases, and further having a coloring group molecule from the 3 end of the nucleic acid analysis probe 2 via a linear substituent. . This one set of probes does not need to be two probes, but may be composed of more than two sets. Even in this case, one set of two probes has a basic configuration, and the operation and effect based on this configuration are the same. Therefore, in the following, a description will be given by taking one probe and two probes as an example.

When the nucleic acid analysis probes 1 and 2 according to the present invention are hybridized with the target polynucleotide, a longer wavelength than the fluorescence generated by the color forming group of the probe 1 or the color forming group of the probe 2 is used. It is characterized by having fluorescence on the side.

Further, in the probe for nucleic acid analysis according to the present invention, when the above-mentioned fluorescence is hybridized with the above-described nucleic acid analysis probes 1 and 2 according to the present invention and the target polynucleotide, the probe 1 An excimer-monofluorescence due to excimer formation between the coloring group and the coloring group of the probe 2. Further, the probe for nucleic acid analysis according to the present invention includes pyrene, naphthalene, anthracene, perylene, stilbene, benzene, toluene, and phenylene as the above-mentioned coloring groups. It is characterized by being selected from the group consisting of Thracene, Diphenirane Thracene, Benzbilene, Benzian Thracene, Tetracene, Fenanthrene, Penyusen, Trifferenylene and Chrysene.

The probe for nucleic acid analysis according to the present invention is characterized in that the coloring group is pyrene.

Further, the probe for nucleic acid analysis according to the present invention includes a length of the above-mentioned chain-like substituent binding the 5 ′ terminal nucleotide of the probe for nucleic acid analysis 1 and the above-mentioned coloring group, and a nucleotide of the 3 ′ terminal of the above probe 2 for nucleic acid analysis. And a length of the chain substituent binding the color-forming group to 3 Å or more and 20 Å or less.

In addition, the nucleic acid analysis probe according to the present invention includes a length of the chain substituent that binds the 5 ′ terminal nucleotide of the nucleic acid analysis probe 1 and the chromophore, The length of the chain substituent that binds the nucleotide and the color-forming group is not less than 5 angstroms and not more than 20 angstroms.

Furthermore, the probe for nucleic acid analysis according to the present invention is characterized in that the 5 ′ terminal nucleotide of the nucleic acid analysis probe 1 and the linear substituent that binds the chromogenic group, or the 3 ′ terminal nucleotide of the nucleic acid analysis probe 2 and the chromogenic the chain substituents attached to the group is chromogenic Kiichi (CH n- (X) k- ( CH 2) characterized in that it is a m-Y- (5 'substituent represented by terminal nucleotide) Here, CONH, NHCO, COO, OCO, 0, S, ΝΗ, are selected from the group consisting of, Υ is selected from the group consisting of 0, S, NH, (POS, Further, n or m represents an integer from 0 to 5, and k represents 0 or 1.

Further, the nucleic acid detection method according to the present invention is a method for detecting a polynucleotide having q base sequences by hybridization with two kinds of nucleic acid analysis probes 1 and two nucleic acid analysis probes 2. A nucleotide and the polynucleic acid Has a complementary nucleotide sequence to r consecutive nucleotides (wherein represents an integer of 1 or more and (q-1) or less) from the 5 'end of leotide, and further from the 5' terminal nucleotide of probe 1 A first nucleic acid analysis probe 1 having a coloring group molecule via a linear substituent, and a complementary nucleotide sequence to a nucleotide sequence between (r + 1) th and qth from the 5 'end of the polynucleotide Further mixing a 3 ′ terminal nucleotide of the nucleic acid analysis probe 2 with a second nucleic acid analysis probe 2 having a coloring group molecule via a linear substituent from the 3 ′ terminal nucleotide; and In this case, the method further comprises a step of measuring the fluorescence ( ¹ ) on the longer wavelength side than the fluorescence by the coloring group of the probe 1 or the coloring group of the probe 2. Further, in the method for detecting nucleic acid according to the present invention, the above-mentioned fluorescence may be obtained by, when the above-mentioned nucleic acid analysis probe 1 and probe 2 and the polynucleotide are hybridized, the coloring group of the above-mentioned probe 1; Characterized by excimer fluorescence due to excimer formation with a color-forming group.

Further, in the nucleic acid detection method according to the present invention, the color-forming group may be pyrene, anthracene, naphthalene, perylene, stilbene, benzene, toluene, phenylanthracene, diphenylanthracene, ben'sopylene, benzuanthracene, tetracene, fenane. It is characterized by being selected from the group consisting of Tren, Pentacene, Trifuenylene and Chrysene.

Further, the nucleic acid detection method according to the present invention is characterized in that the coloring group is biylene.

Further, the nucleic acid detection method according to the present invention comprises: a length of the chain substituent that binds the 5′-terminal nucleotide of the nucleic acid analysis probe 1 to the chromophore; and a 3′-terminal nucleotide of the nucleic acid analysis probe 2. And a length of the chain-like substituent connecting the above-mentioned coloring group to the color-forming group is not less than 3 Å and not more than 20 Å.

Further, the method for detecting nucleic acid according to the present invention comprises the step of: The length of the chain substituent that binds leotide to the color-forming group and the length of the chain substituent that bonds the 3′-terminal nucleotide of the probe 2 for nucleic acid analysis to the color-forming group are 5 Å or more. It is characterized by being less than 20 angstroms.

Further, the nucleic acid detection method according to the present invention may further comprise: the above-mentioned chain-like substituent binding the 5′-terminal nucleotide of the above-mentioned nucleic acid analysis probe 1 and the above-mentioned coloring group; And the above-mentioned chain-like substituent that binds to the above-mentioned coloring group is a substituent represented by a coloring group 1 (CH ₂ ) n- (X) k— (CH,) m— Y— (5 ′ terminal nucleotide) It is characterized by the following. Where X is selected from the group consisting of CONH, NHCO, COO, 0C0, 0, S, NH, Y is selected from the group consisting of 0, S, NH, and n or m is 0 to 5 Represents an integer up to and k represents 0 or 1.

More specifically, the nucleic acid detection probe according to the present invention is a polynucleotide, and the target polynucleotide is hybridized with two types of nucleic acid detection probes 1 and 2 according to the present invention. It strictly recognizes the nucleotide sequence.

Furthermore, the two types of probe polynucleotides according to the present invention have excimer (or excibrex) -forming chromophores such as pyrene at the 5 ′ and 3 ′, respectively, and the target polynucleotide and the target polynucleotide The complementary hybridization of the polynucleotides of the probe according to the present invention causes the color-forming group molecules to take a spatially close positional relationship, and with the decrease in fluorescence inherent to the monomer of the color-forming group. The fluorescence shifted to the long wavelength side can be observed.

Furthermore, if the two chromophores are in a spatially favorable positional relationship, a strong excimer or exciplex can be formed, accompanied by a significant decrease in the fluorescence inherent to the chromophore monomers. Strong excimer or exciplex fluorescence shifted to the longer wavelength side can be observed. Thus, identification and detection of the target polynucleotide can be performed by measuring the above-mentioned shifted fluorescence, excimer, or exciplex fluorescence. Alternatively, this can be achieved by measuring the decrease in monomer-fluorescence. Hereinafter, preferred embodiments of the present invention will be described.

(Target nucleic acid (target nucleic acid, target polynucleotide))

The target nucleic acid that can be detected using the nucleic acid detection probe according to the present invention is not particularly limited, and can be hybridized by a generally known method, for example, DNA, RNA (tRNA, mRNA, rRNA). RNA), a synthetic oligonucleotide, a synthetic polynucleotide, a synthetic deoxyoligonucleotide, a synthetic deoxypolynucleotide, or a heteropolymer of deoxyribonucleotide and liponucleotide. Furthermore, it is necessary that the specific portion of the nucleotide sequence of the target nucleic acid is known in advance, and based on this nucleotide sequence information, a probe that is completely complementary to the nucleic acid is prepared and hybridized. .

For the purpose, as a method for knowing the base sequence of the target nucleic acid, a known base sequence determination method can be used (for example, the Sanger method (dideoxy-mediated chain-termination method for DNA sequencing)).

(Probe for nucleic acid detection)

The polynucleotide as a probe for nucleic acid analysis according to the present invention is one in which one set of two types of polynucleotides completely complementarily hybridizes to a specific portion of the polynucleotide to be detected.

That is, as shown in FIG. 2, the two types of polynucleotides related to the present invention have a nucleotide sequence in which probe 1 and probe 2 completely complementarily hybridize with the nucleotide sequence of the target polynucleotide. Each has a base sequence so as to have It is possible to determine the number of base sequences of any number of probes 1 from the 5 'end of a specific portion of the target nucleic acid, and the base sequence of probe 2 is automatically determined accordingly.

However, if the number of bases of either probe is too short, it will be difficult to recognize a sufficient complementary base sequence. Therefore, the preferable number of bases has a range, and therefore, the number of base sequences of the probe 2 or the probe 1 is preferably both 8 or more.

Furthermore, in the present invention, the method for synthesizing the nucleotide sequence necessary for the probe 1 or 2 is not particularly limited, and a general nucleotide modification method (for example, Handbook of Fluorescent Probes and Research Chemicals, 5th ed, 1992-1994, by RPHaugland, Molecular Probes, Inc) or an automated synthesis method (for example, the method described in Oligogon ucleotides and Analogues A Practical Approach, ed by F. Eckstein, IRL Press). is there. It is also possible to introduce a plastid into a natural oligonucleotide before use.

The resulting polynucleotide can be purified, for example, by reversed-phase high-performance liquid chromatography.

(Chain substituent (phosphorus))

In addition, the polynucleotides of these two types of probes according to the present invention may have a chain of an appropriate length in order to provide a labeling group (color-forming group molecule, chromophore, fluorophore) at the 5 ′ or 3 ′ end. The probe is linked to the probe base sequence via a substituent (linker).

When these two probe polynucleotides hybridize to the target polynucleotide, the two chromophore molecules from each of the probe polynucleotides are spatially arranged so as to allow a closer position to the linker. . Therefore, the length from the 5 ′ or 3 ′ end to the coloring group is extremely small in the present invention. It can be adjusted according to the type and length of the linker. In particular, it is clear from the findings of the inventors that the length of the linker greatly affects the detection sensitivity in the present invention. FIG. 6 shows that excimer formation based on birene-pyrene is greatly affected by the length of the linker. Therefore, in the present invention, the length of the linker is a measure for optimization, and the length of the linear substituent (linker) is defined as the length of the C-C from the probe polynucleotide to the chromogenic group molecule. , C-1 0, CN, N—N, C—S, P—0, etc. When all the single covalent bonds are of the same length (1.4 Å), this is calculated from the number of bonds. Means

For example, from the 5 'carbon of the 5' terminal nucleotide, the following chain substituent (linker) (5'-C) — 0—P—S_CH—C0NH—CH′—chromophore molecule

When it is combined with a chromophore, the result is 1.4 x 8 = 11.2 angstroms.

Further, for example, 3 carbon '3' terminal nucleotide, the following chain substituent (Li down force one) (3 '-C) one _{NH- NH-CO- CH2- CH2- CH 2} - CH2- chromophore When combined with a chromophoric group in the molecule, the result is 1.4x8 = 11.2 Angstrom

As shown in Fig. 6, in order for the color-forming group molecules to be spatially close to each other and the interaction between the molecules to be recognized, it is effective if the linker is too short or too long. It is thought that it is difficult to take a proper spatial arrangement.

For example, according to the findings of the present inventors, in the case of a bienyl group as a color-forming group, it is preferably located in a space at least 3 angstroms away from the 5 'or 3' carbon, and more preferably from 5 angstroms to 20 angstroms. Was found to be good.

Further, when the above-mentioned chain substituent (linker) is 3 Å or less, the proximity of the color-forming groups becomes sterically impossible. In this case, the formation of excimer and the like is not possible. It is considered that shimmering fluorescence or the like cannot substantially occur.

It is also effective to study using a C PK molecular model, and it is presumed that the length is preferably about 3 Å or more, and more preferably 20 Å or less. More preferably, it is 5 angstrom or more and 15 angstrom or less. More specifically, according to the findings of the present inventors, it was found by actual measurement of excimer fluorescence that the range of 5 to 10 angstroms is a preferable range when the virenyl group is a coloring group. That is, the present invention does not include the case where a bienylmethyl group is bonded to the 5 ′ or 3 ′ carbon by an ether bond (4.2 angstrom).

In the present invention, the type of the linker and the synthesis method are not particularly limited as long as the linker having the above length can be obtained. For example, a methylene group, an amide group, an ester group, an ether group, a thiophosphate, or a combination thereof can be suitably used. It is an easy choice for a person skilled in the art to determine an appropriate linker-binding mode in consideration of chemical stability, thermal stability, suitability as a labeling group, and the like.

(Color-forming group, labeling group, chromophore, fluorophore)

The coloring group (or labeling group) usable in the present invention means a group containing a chromophore in at least a part thereof, and is not particularly limited as long as it easily forms an excimer or an exciplex. Various kinds of coloring groups can be introduced by a general synthesis method.

In particular, the chromophores include pyrene, naphthylene, anthracene, perylene, stilbene, benzene, toluene, phenylanthracene, diphenylanthracene, benzene, pentapyrene, benzuanthracene, tetracene, phenanthrene, pengysen, triffeenylene, It is selected from the group consisting of chrysene, and aromatic chromophores can be suitably used, and more preferably, a chromophore having a pyrene skeleton can be used. In the present invention, preferably, the coloring groups of the two types of probes are the same. That is, it is not a so-called donor: acceptor type in the present invention.

As will be explained later, the long wavelength shifted fluorescence according to the present invention is therefore not of the electron transfer type. That is, in the present invention, excimer or exciplex fluorescence due to excimer or exciplex formation by the same chromophore molecule can be observed, and two types based on the hybridization described above are used. Since the color-forming groups are spatially close to each other and generate the above-mentioned fluorescence, only those which have been correctly hybridized to the target nucleic acid can be detected. Therefore, even when the probe according to the present invention coexists, it is possible to detect only hybridized ones without interference from the fluorescence derived from the chromogenic monomer itself. Become.

(Hyperdaidise)

The method for hybridizing with the target polynucleotide using the polynucleotides of the two nucleic acid detection probes according to the present invention is not particularly limited.

Conventional hybridization conditions can be suitably used (for example, the method described in Molecular Cloning A Laboratory manual 2nd. Ed, J. sambrook et al., Cold Spring Harbor Laboratory Press, 1989).

For example, by mixing two probes and a target nucleic acid in a solution at 25 ° C., hybridization can be suitably performed. Alternatively, the reaction may be carried out at a higher temperature (for example, at a temperature lower by 10 ° C. than the melting temperature), followed by annealing to return to room temperature, thereby performing hybridization.

Furthermore, in the present invention, identification or quantification of a target polynucleotide can be performed after hybridization without washing or other means. In the present invention, the detection method is not particularly limited as long as it is a means for measuring the fluorescence after hybridization. For example, a commercially available fluorometer Thus, the measurement can be suitably performed.

As shown in Fig. 4 etc., in addition to the fluorescence of the chromophore itself (in this case, bilen) (beak wavelength is 38 O nm), the excimer fluorescence shifted to the longer wavelength side (the beak wavelength is about 5 (0 nm) can be measured. Of course, the excimer beak is not observed at all before the hybridization. Therefore, the quantification of the hybridized target is quite easy, and by preparing an accurate calibration curve, the target nucleic acid concentration can be calibrated by measuring the fluorescence intensity as shown in Fig. 5. it can. The experimental data of FIG. 5 using a normal fluorometer does not indicate the detection limit of the present invention, and it is possible to detect with higher sensitivity by improving the sensitivity of the fluorometer. Optimization of this measurement system is a matter that can be easily selected. However, FIG. 6 shows that detection of at least several nM is possible.

In particular, the analytical probe according to the present invention may be used in excess, and the above operation may be performed without any interference with the fluorescence based on the chromophore of the excess probe that has not been hybridized. It is possible.

(Rapid extraction of point-mutated nucleic acids)

As described above, the principle of the detection according to the present invention is that, as described above, the two probes correctly hybridize to the target nucleic acid, thereby generating strong excimer fluorescence, and measuring the target nucleic acid to measure the target nucleic acid. It is for detection and quantification. Therefore, even if there is a nucleobase that cannot form a base pair between the two probe ends to which the labeling group is bonded, use a designed probe by excluding that portion from the base sequence of the probe. Accordingly, the present invention can be used for a highly sensitive and selective detection method for an evening-get nucleic acid, ie, a point-mutated nucleic acid, which differs only in their base sequences. This possibility is shown by the fact that the presence of one or two nucleobases between two probes, as shown in FIG. Then, it becomes possible to observe the identification of the point mutation from the result (Fig. 8). Hereinafter, the present invention will be described in detail with reference to Examples. However, the invention is not limited to this embodiment.

A liponucleotide was used for the 3′-terminal nucleotide of probe 2 (compound 3) described below, and deoxyribonucleotide was used for all others.

(Synthesis of Oligonucleotides)

The oligonucleotide 32 -mer chosen as the model to be detected has the following sequence: 5′-AGAGGGCACGGATACCGCGAGGTGGAGCGAAT-3 ′ (compound 1). A nucleotide having a nucleotide sequence complementary to the 16 nucleotide sequences from the 5 'end of the subject was used as a probe 1 for nucleic acid detection: 3'-TCTCCCGTGCCTATGG-5' (compound 2). Further, a nucleotide having a complementary nucleotide sequence to the 17th to 32nd nucleotide sequence from the 5 'end of the oligo nucleotide 32 -mer (compound 1) was used as a nucleic acid detection probe 2: 3' -(C) GCTCCACCTCGCTTA-5 '(compound 3) (however, only (C) is a ribonucleotide). All of these were synthesized using an automatic synthesizer manufactured by Millipore Limited in accordance with the solid phase phosphoramidite synthesis (SO LID STATE PHOSPHORAMIDITE TECHNIQUE) method.

The oligonucleotides 32 -mer (compound 1) and two kinds of oligonucleotides 16 -mer (compound 2) and (compound 3) obtained therefrom were subjected to reversed-phase high-performance liquid chromatography (column : PepRPCTM 4m HR5 / 5 manufactured by Pharmacia Fine Chemicals, gradient elution solvent system acetonitrile / 0.1M ammonium acetate mixed solvent, detection wavelength 260 nm).

Furthermore, each was desalted by gel filtration and concentrated to dryness by freeze-drying.

(5 'end of nucleic acid detection probe 1 (3'-TCTCCCGTGCCTATGG-5') (compound 2) -The oligonucleotide of probe 1 (compound 2) obtained above (molecular weight 4904.2 1, extinction coefficient £ l39.9mmol— 'liter · cm-) (Czworkowski. J., et. Al., Biochemistry, 30, p. 4821, 1991) and introduced in the following procedure.

That is,

(i) 2 ml of (compound 2), 2.5 ml of a solution of 20 mM MgCl ₂ and 140 mM Tris-HCl (pH 7.6) containing 0.2 M KC1, 2.135 ml of water, 0.05 ml of 500 mM thiothreitol, and lOOmM adenosine one 5 '- 0- (3' - thio triforine scan Fei g) (lithium salt, base one Ringer Mannheim) 0.5 ml and, T ₄ polynucleotide force rice Ichisu solution (pol ynucleotide kinase solution, Takara) 0.04 ml And leave at 36 ° C for 3 hours protected from light.

(ii) Further, after adding 2.5 ml of 2M NaCl and 2.5 ml of water, a mixed solution of chloroform and methanol at a volume ratio of 1: 1 was added, followed by centrifugation to collect an aqueous phase.

(iii) 10 ml of chloroform was added to the obtained aqueous phase, and the aqueous phase was recovered after centrifugation. The deoxyoligonucleotide was precipitated by ethanol, concentrated, and equilibrated with 25 mM HEPES / NaOH (pH 7.4) containing 0.5 mM NaCl and 10 mM mercaptoethanol (Sephadex G-25, Pharmaciafu). The solvent was exchanged using a column (Ein Chemicals).

(iv) After deoxyoligonucleotides are dried by ethanol precipitation and centrifugal concentration, the following solvents are added sequentially. That is, 4 ml of 50 mM bicine / K0H (pH 8.4), 5 ml of dimethylformamide (DMF), and N- (topyrenylmethyl) iodide acetate (N- (l-pyrenylmethyl) 1.0 ml of a DMF solution of iodoacetamide (Molecular Probes) was added and stirred at room temperature for 4 hours.

(V) Excess reaction solution was removed by ethanol precipitation, and (compound 4) was obtained by centrifugal concentration. Figure 9 shows the structure of (Compound 4). (vi) ODS reverse phase high performance liquid chromatography was used for further purification. The column used was a TSKgel OligoDNA RP (Tosoichi) column (21.5 pictures, 15 cm).

For elution, a mixture of acetonitrile and 0.1 M ammonium acetate was eluted with the following concentration gradient (elution rate: lml / min, temperature: room temperature). After 5 minutes at 5:95 for the first 5 minutes, a gradient from 5:95 to 40:60 in 175 minutes.

(vii) Centrifuge the obtained sample at room temperature, dissolve the final product in 0.01M Tris-HCl buffer (pH7.5) containing 3.0mM EDTA and 0.1M NaCI, and freeze at 185 ° C. saved.

(Introduction of pyrene chromophore to 3 'end of nucleic acid detection probe 2 (3'-(OGCTCCACCTCGCTTA-5 ') (Compound 3) Part 1)

A pyrene group was added to the oligonucleotide (compound 3) (molecular weight 4849.18, extinction coefficient e 136.4 mm 1 · liter · cm-1) of probe 2 obtained above, and the method of Reins, Cantor, et al. (Koenig, P , Reins, SA, Cantor, CR (1977) Pyrene Derivatives as Fluore scent Probes of Conformation Near the 3 'Termini of Polyribonucleotides ",

Biopolymers 16, 2231-2242) was modified and introduced as follows.

(i) Dissolve 45.5 μg of the oligonucleotide (compound 3) in 0.5 ml of 0.05 M acetate buffer (pH 5.6).

(ii) Add 2.0 ml of 0.05M acetate buffer (ρΗ5.6) containing HOmg NaI04 and 7.5M urea, and leave it at room temperature for 45 minutes in the dark.

(iii) Add 0.5 ml of KC1 and leave at 4 ° C to precipitate excess NaI04.

(iv) After removing the precipitate by centrifugation, the oligonucleotide is precipitated by ethanol precipitation.

(V) The precipitate was dissolved in a small amount of 0.05 M acetate buffer (pH 5.6) containing 3 mM EDTA, and then desalted using a Sephadex G-25 column equilibrated with the same buffer.

(vi) Collect the oligonucleotide fractions based on the absorbance at 280 nm and concentrate them using an ethanol precipitation procedure. (vii) To the precipitate, added 0.05> 1 acetate buffer (115.6) of 3.01111, and further added 3.0 ml of dimethylsulfoxide (DMSO) in which 3 mg of 1-pyrenebutyric acid hydrazide was dissolved. The mixture was left for 2 hours in the light shielded at ° C.

(viii) After addition of 0.3 ml of 2M KC1, an excess of ethanol was added to precipitate the oligonucleotide.

(ix) After centrifugation, 1.0 ml of a 0.01 M Tris-HCl (pH 7.5) buffer containing 3.0 mM EDTA sodium salt and 0.1 M KC1 was added to the precipitate to redissolve the precipitate.

(X) Finally, NaBH3CN (sodium cyanoborohydride) was added, and the reduction reaction was performed at room temperature for 1 hour. The amount of addition was about 320 times the molar amount of the probe.

(xi) After that, the ethanol precipitation operation was repeated several times, and after confirming that the fluorescence derived from the birene in the supernatant after centrifugation was completely eliminated, the final precipitate was dried and solidified by centrifugation to obtain (Compound 5). Was. Figure 10 shows the structure of (Compound 5).

(xii) Purification was performed by 0DS reversed-phase high-performance liquid chromatography. The column used was TS Kgel OligoDNA RP (manufactured by Tosoh I) (21.5 mm x 5 cm). The solvent used was a gradient of a mixed solution of acetonitrile and 0.1 M ammonium acetate.

(xiii) The obtained (compound 5) is concentrated by centrifugation at room temperature, dissolved in a 0.01 M Tris-HCl buffer (pH 7.6) containing 3.0 mM EDTA sodium salt and 0.1 M NaCl, and frozen at 85 ° C. saved.

(Introduction of a pyrene coloring group at the 3 'end of nucleic acid detection probe 2 (3'-(OGCTCCACCTCGCTTA-5 ') (Compound 3) Part 2)

The following method was also used to introduce a pyrene coloring group at the 3 'end of the nucleic acid detection probe 2 (compound 3). In this method, the method of BPGottikh et al. (Gottikh, BP, Krayev sky, AA, Tarussova, NB, Tsilevich, T., Tetrahedron, 26, 4419-4433 (1970)) was used with some modifications. . Here, this method is called CDI (carbonyldiimidazol) method. (i) Prepare a 1.2M CDI solution in DMF.

(ii) To 0.1 ml of this solution, add 0.1 ml of 0.4 M 1-Pyrenebutanoic acid DMF solution and react at room temperature for about 30 minutes.

^) Add a 16-1116 aqueous solution with a concentration of 40011111001 / 0.5101 to the above solution and incubate at room temperature under dark conditions for 3 hours.

(iv) Remove the solvent using a centrifugal evaporator.

(V) To this residue, water and chloroform are sequentially added, and solvent extraction is performed.

(vi) Obtain a water fraction, add form again, and perform solvent extraction.

(vii) The solvent of the obtained water fraction is removed using a centrifugal evaporator.

(viii) Purification was performed by adding a small amount of water and using an ODS reverse-phase high-performance liquid chromatograph. As the column, a TSKgel OligoDNA RP (Tosoichi) column (21.5 mm × 5 cm) was used. A mixture of acetonitrile and 0.1 M ammonium acetate was eluted with the following concentration gradient (elution rate: lml / min, temperature: room temperature). After 5 minutes at 5:95, gradient from 5:95 to 40:60 in 175 minutes.

(ix) The obtained sample (Pyrenebutanoic acid-introduced 16-mer) (compound 6) is dissolved in IM phosphate buffer (pH7) and stored at -85 ° C. Figure 11 shows the structure of (Compound 6).

(Basic hybrid operation)

20% (volume ratio) In lOmM phosphate buffer solution (PH7.0) containing dimethylformamide (DMF) and 0.2M NaCl, the target oligonucleotide (compound 1), probe oligonucleotide (compound 4) and ( Compound 5) was mixed with about 66 nM each and hybridized at 25 ° C. By monitoring the absorbance (A260) of the nucleic acid with respect to the temperature of this solution, that is, the deep color effect, it was confirmed that the hybridisation completely formed complementary base pairs at a temperature of 25 ° C or less. I found it. (Confirmation of excimer formation)

The fluorescent spectrum was measured under the basic hybridization conditions described above. As shown in Fig. 4, the characteristic fluorescence of the pyrene monomer was observed around 400 ran, and at the same time, a broad fluorescence band was observed around 500 nm. This band with a wavelength around 500 nm is observed only when the above three types of oligonucleotides are present, and resembles the fluorescence spectrum of a conventionally known bilen excimer. Thus, it is thought to be due to excimer monofluorescence caused by the bimer dimer (Birks, JB Chri stophorous, LG (1963)), Spectrochim. Acta 19, 401-41

0) o

If the fluorescence band at 495 nm is due to excimer fluorescence, its intensity should be accompanied by a decrease in the fluorescence of birene itself.Accordingly, the intensity ratio of the two fluorescence bands described above will change due to the change in the concentration of the target oligonucleotide. It was found to change complementarily (Fig. 4).

From the above results, it was concluded that the fluorescence band near 500 nm was excimer fluorescence.

(Possibility of quantitative analysis of evening nucleic acid)

Figure 5 shows the fluorescence around 500 nm when various concentrations of the target oligonucleotide were added to a mixture of equal amounts of the probe (compound 4) and (compound 5) for detecting excess nucleic acid. It shows the relative intensity of the band. By using such a calibration curve, it is possible to quantify the target oligonucleotide.

(Excimer fluorescence intensity dependence based on linker length)

Figure 6 shows the fluorescence spectrum obtained when the equimolar pyrenebutyric acid-introduced 16-mer probe and the yrenealkyl iodoacetamide-introduced 16-mer were hybridized to the target nucleic acid 32-iner. It shows. The term "pyrenealkyl iodoacetamide" as used herein means that the linker portion of pyrenemethyl iodoacetamide (ie, the alkyl chain portion) has a different length. However, the synthesis method is exactly the same as for pyrenemethyl iodoacetamide.

As is evident from Fig. 6, even if one probe is the same (in this case, pyrene butan oic acid-introduced 16-mer), it is significant only by changing the length of the linker part of the other probe by one methylene chain. In addition, the quantum yield of excimer fluorescence is affected. Under these experimental conditions, pyrenemethyl iodoacetamide-introduced 16-mer showed the highest quantum yield, and the linker could be longer or shorter.

This suggests that the optimal length of the linker is 11.4 Å from the C at the 5 'end of Deoxyribose. The shortest PIA is 9.8 Angstrom and the longest PPIA is 12.6 Angstrom.

(Detection of point mutation target nucleic acid)

Furthermore, the present inventors have studied a hybridization in which the above-described two probes according to the present invention are not completely continuous with the target nucleic acid. In other words, as shown in Fig. 7, when hybrids are formed, if the distance between two probes is 1-2 nucleotides from the target nucleic acid (Fig. 8), excimer formation is significantly suppressed. It was confirmed. That is, it is understood that the necessity that two probes are continuously arranged for the target nucleic acid is important for excimer formation.

This fact means that wild-type and point-mutated genes can be distinguished and identified in a homogeneous solution by designing two probes and performing hybridization so that the point-mutated portion is located in the middle. .

Here, we focus on one or two thimine deoxyribonucleotides added in the middle of the 32-mer described earlier (that is, 33 and 34 mer). this Due to this, the ends adjacent to each other (one professional

The distance between the 5 'end of the probe and the other 3' end is likely to increase (see Figure 8). As a result, it is thought that the optimal conformation for excimer formation cannot be obtained. Industrial applicability

Therefore, according to the present invention, there is provided a probe for detecting a nucleic acid which is non-radioactive and is based on hybridization, and which is highly sensitive and capable of recognizing a difference of one base. After the operation, the complex hybridized with the probe can be specifically detected without washing and removing the probe coexisting in excess.

Claims

The scope of the claims

1. The first nucleic acid analysis probe 1 or the second nucleic acid analysis probe 2 for detecting a evening polynucleotide having q base sequences, the base of the first nucleic acid analysis probe 1 The sequence is a complementary nucleotide sequence to r consecutive nucleotide sequences (r is an integer of 1 or more and (q-l) or less) continuous from the 5 'end of the above-mentioned polynucleotide. At the 5 'end has a coloring group via a chain substituent,

The base sequence of the second nucleic acid analysis probe 2 is a base sequence complementary to the base sequence between (Γ + l) th and qth from the 5 ′ end of the target polynucleotide, and Has a coloring group from the 3 ′ end of the probe 2 via a chain substituent,

A nucleic acid for nucleic acid analysis, which emits fluorescence having a longer wavelength than fluorescence based on a coloring group of the probe 1 or probe 2 during hybridization of the probe 1 and the probe 2 with the target polynucleotide. probe.

2. The nucleic acid according to claim 1, wherein the long-wavelength fluorescence is exciplex fluorescence based on exciplex formation of a color-forming group of the probe 1 and a color-forming group of the probe 2. Analytical probe.

3. The probe for nucleic acid analysis according to claim 1, wherein the long-wavelength fluorescence is excimer fluorescence based on excimer formation between the coloring group of the probe 1 and the coloring group of the probe 2.

4. The coloring group bonded to the probe 1 or probe 2 is pyrene, naphthalene, anthracene, perylene, stilbene, benzene, toluene, phenylanthracene, diphenylanthracene, benzopyrene, benzuanthracene, tetracene, phenanthrene. And at least one coloring group selected from the group consisting of pentacene, trifluorocene, and chrysene. The probe for nucleic acid analysis according to 1.

5. The probe according to claim 4, wherein the coloring group is pyrene.

6. The probe for nucleic acid analysis according to claim 1, wherein the length of the chain substituent bonded to the probe 1 or the probe 2 is 3 Å or more and 20 Å or less. .

7. The probe for nucleic acid analysis according to claim 6, wherein the length of the chain substituent is not less than 5 angstroms and not more than 20 angstroms.

8 The linear substituent bonded to the nucleic acid analysis probe 1 or the linear substituent bonded to the nucleic acid analysis probe 2 is

The substituent represented by the coloring group 1 (CH ₂ ) _n- (X) _k- (CH ₂ ), „Y— (5 ′ terminal nucleotide) is represented by CONH, NHCO, COO, 0C0, 0, S, Selected from the group consisting of H, Y is selected from the group consisting of 0, S, NH, (PO) S, n or m represents an integer from 0 to 5, and k represents 0 or 1. 4. The probe for nucleic acid analysis according to claim 1, wherein:

A method for detecting a target polynucleotide having 9 q base sequences based on hybridization with a first nucleic acid analysis probe 1 and a second nucleic acid analysis probe 2;

The evening gate polynucleotide;

The probe 1 has a complementary nucleotide sequence to r consecutive nucleotides (r is an integer of 1 or more and (q-1) or less) from the 5 ′ terminal nucleotide of the evening-get polynucleotide, and further comprises the probe 1 A first nucleic acid analysis probe 1 having a coloring group from the 5 'terminal nucleotide via a linear substituent,

The target polynucleotide has a complementary base sequence to the base sequence between the r-th and q-th base nucleotides from the 5′-end nucleotide of the target polynucleotide, and further has a base sequence from the 3 ′ end of the nucleic acid analysis probe 2 described above. Second having a coloring group via a chain substituent Mixing with the nucleic acid analysis probe 2 of the above;

A step of measuring fluorescence on a longer wavelength side than fluorescence by the coloring group of the probe 1 or the coloring group of the probe 2 during the hybridization by the mixing;

A method for detecting a target polynucleotide, comprising at least:

10 When the long-wavelength-side fluorescence is hybridized with the nucleic acid analysis probes 1 and 2, and the overnight polynucleotide, the color-forming group of the probe 1 and the probe 10. The detection method according to claim 9, wherein the second coloring group is exciplex fluorescence generated by exciplex formation.

11 1 When the fluorescence on the long wavelength side is used for hybridization of the nucleic acid analysis probes 1 and 2 and the target polynucleotide, the color-forming group of the probe 1 and the probe 2 10. The detection method according to claim 9, wherein the color-forming group is excimer fluorescence due to excimer formation.

1 2 The above coloring groups are pyrene, naphthylene, anthracene, perylene, stilbene, benzene, toluene, phenylanthracene, diphenylanthracene, benzovirene, benzuanthracene, tetracene, phenanthrene, penycene, triene 12. The detection method according to claim 9, wherein the detection method is selected from the group consisting of phenylene and chrysene.

13. The detection method according to claim 12, wherein the coloring group is pyrene. 14.The detection method according to claim 9, wherein the length of the chain substituent bonded to the probe 1 and the probe 2 is 3 Å or more and 20 Å or less. .

15. The detection method according to claim 14, wherein the length of the chain substituent is not less than 5 angstroms and not more than 20 angstroms.

16 The above-mentioned chain substituent of the nucleic acid analysis probe 1 is

Chromophore (CH ₂ ), — (X) _k- (CH ₂ ) _m -Y-(5 'terminal nucleotide) (X is selected from the group consisting of CONH, NHCO, COO, OC〇, 〇, S, NH, Y is selected from the group consisting of 0, S, NH, (PO) S, 12. The detection method according to claim 9, wherein n or m represents an integer from 0 to 5, and k represents 0 or 1.).