JP2012170373A - Hybridization agent for in-stem beacon-type probe and use thereof - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a hybridization agent for an in-stem beacon-type probe securing hybridization potency of the in-stem beacon-type probe with a target nucleic acid, and improving detection sensitivity of the target nucleic acid.SOLUTION: There is provided the hybridization agent for the in-stem beacon-type probe containing a cationic polymer as an active component, having a loop region and a stem region, and containing a fluorescent dye and a quencher dye in the stem region.

Description

  The present invention relates to a hybridization agent for an in-stem beacon type probe and use thereof.

  Conventionally, a molecular beacon includes a loop region that is a sequence recognition site and a stem region that is a self-complementary strand site, and is used as a probe for detecting a target nucleic acid. The molecular beacon has a fluorescent dye and a quenching dye at the end of the stem. At the time of forming the stem, the fluorescent dye is quenched, and recognizes the base sequence of the target nucleic acid at the loop site, so Upon hybridization, when the stem is opened, the action of the quenching dye on the fluorescent dye is released and emits fluorescence.

  Such molecular beacons are expected to be highly versatile due to the high degree of freedom in designing the sequence recognition site of the loop site and the intrinsic extinction mechanism. On the other hand, there are cases where the quenching effect is insufficient, and the introduction site of the fluorescent dye is limited to the end.

  Therefore, the present inventors have already promoted quenching by stabilizing the stem region at the time of stem duplex formation by introducing a fluorescent dye and a quenching dye as a pseudobase using a specific skeleton in the stem region. A molecular beacon that can lower the background has also been proposed (Patent Document 1). According to this molecular beacon, it is possible to enhance the light emission by promoting the quenching by selecting the introduction form of the fluorescent dye and the quenching dye.

  On the other hand, it is known that cationic polymers promote the formation of DNA double strands and promote the strand exchange reaction of double-stranded DNA (Patent Document 2).

WO2010 / 001902 pamphlet JP 2008-278779 A

  By introducing a plurality of pairs of fluorescent dyes and quenching dyes into the stem region of the in-stem beacon type probe, it can be expected to promote quenching while enhancing fluorescence. However, according to the study by the present inventors, when such sensitivity optimization is performed, even if the target nucleic acid is present, the stem duplex of the beacon probe is difficult to open, and the opening speed is slow. I found out there was a problem. That is, when trying to detect the target nucleic acid with high sensitivity, there is a problem that it cannot be hybridized with the target nucleic acid. In particular, in the case of a stem duplex employing the backbone structure disclosed in Patent Document 1 and forming a stack structure of a fluorescent dye and a quenching dye, the target nucleic acid is improved as a result of improvement for improving sensitivity. In some cases, no hybridization occurred.

  It is described that the cationic polymer disclosed in Patent Document 2 promotes hybridization and strand exchange in DNA single strands and duplexes. However, no report has been applied to the beacon type probe, and no report has been applied to the in-stem beacon type probe. Further, even the skilled person could not predict the action of the cationic polymer in the in-stem beacon type probe obtained by using a specific backbone during the formation of the stem duplex.

  Therefore, the disclosure of the present specification is provided to solve the above-described problem in the in-stem beacon type probe. That is, the disclosure of the present specification discloses a hybridization agent for an in-stem beacon probe that can ensure the hybridization ability of the in-stem beacon probe with the target nucleic acid and improve the detection sensitivity of the target nucleic acid. And provide its use.

  The in-stem beacon type probe forms a hybridization state in which a stem duplex is formed by base pairing in the stem region when the target nucleic acid is not present, and bases in the loop region and / or the stem region when the target nucleic acid is present. A hybridization state in which a duplex is formed with the target nucleic acid by pairing is formed. That is, in any state, a base-pairing state with a complementary strand is formed. In such an in-stem beacon type probe, in order to promote the base pairing state in the state where the stem is opened in the presence of the target nucleic acid while stabilizing the stem duplex in the absence of the target nucleic acid, We thought that some kind of additive was necessary to bias the equilibrium of the state to the base pairing state at the time of opening the stem duplex.

  As a result, the present inventors surprisingly promoted and stabilized quenching by allowing the cationic polymer to act on the molecular beacon in the absence of the target nucleic acid, and converting the cationic polymer to the molecular beacon in the presence of the target nucleic acid. As a result, it was found that hybridization with the target nucleic acid was further promoted and stabilized, and the present invention was completed. The present specification provides the following disclosure.

(1) Cationic polymer as an active ingredient,
A hybridization agent for an in-stem beacon type probe, which is a beacon type probe having a loop region and a stem region, and having a fluorescent dye and a quenching dye in the stem region.
(2) The hybridization agent according to (1), wherein each of the fluorescent dye and the quenching dye is provided in the stem region of the in-stem beacon probe as the following unit.
(In the formula, X represents a fluorescent dye, R 1 represents an alkylene chain having 2 or 3 carbon atoms and may be substituted, and R 2 has 0 to 2 carbon atoms and is substituted. Represents an alkylene chain which may be substituted, and Z represents a direct bond or a linking group.)
(In the formula, Y represents a quenching dye, R 1 represents an alkylene chain having 2 or 3 carbon atoms, which may be substituted, and R 2 has 0 to 2 carbon atoms and is substituted. Represents an alkylene chain which may be substituted, and Z represents a direct bond or a linking group.)
(3) The hybridization according to (1) or (2), wherein the in-stem beacon type probe has two or three combinations of one each of the fluorescent dye and the quenching dye in the stem region. Agent.
(4) The hybridization agent according to (3), wherein the in-stem beacon type probe has four or more combinations in the stem region.
(5) The hybridization according to any one of (1) to (4), wherein the cationic polymer is a copolymer composed of a polymer chain having a cationic group and a hydrophilic polymer chain. Agent.
(6) The hybridization agent according to (5), wherein the polymer chain having a cationic group is polylysine or polyallylamine.
(7) The hybridization agent according to (5) or (6), wherein the hydrophilic polymer chain is dextran or polyethylene glycol.
(8) The cationic polymer according to any one of (1) to (7), wherein the cationic polymer includes at least one of α-PLL-g-Dex, ε-PLL-g-Dex, and PAA-g-Dex. Hybridization agents.
(9) The hybridization agent according to any one of (1) to (8), further comprising the in-stem beacon type probe.
(10) The hybridization agent according to any one of (1) to (8),
A beacon type probe having a loop region and a stem region, the in-stem beacon type probe having a fluorescent dye and a quenching dye in the stem region;
A hybridization kit comprising:
(11) The hybridization kit according to (10), wherein one or more of the in-stem beacon probes are immobilized on a solid phase carrier.
(12) A hybridization method between an in-stem beacon type probe and a target nucleic acid,
In the presence of the hybridization agent according to any one of (1) to (8), the target nucleic acid, a loop region, and a stem region are included, and a fluorescent dye and a quenching dye are included in the stem region. And a step of contacting an in-stem beacon type probe under a condition capable of hybridization to obtain a signal based on the fluorescent dye of the in-stem beacon type probe in the presence of the target nucleic acid.
(13) The method according to (12), further comprising a step of acquiring a signal based on the fluorescent dye of the in-stem beacon probe in the presence of the hybridization agent in the absence of the target nucleic acid. the method of.
(14) The method according to (12) or (13), wherein the in-stem beacon probe has two or three combinations of one of the fluorescent dye and the quenching dye in the stem region. .
(15) The method according to (12) or (13), wherein the in-stem beacon type probe has four or more combinations in the stem region.

It is a figure which shows the time change of fluorescence intensity accompanying target nucleic acid (Surv-2) addition. It is a figure which shows spot arrangement | positioning of the in-stem beacon type probe on the board | substrate used in Example 4. FIG. It is a figure which shows the fluorescence intensity level of the fluorescent dye of the in-stem beacon type probe on the substrate in the presence and absence of the cationic polymer. It is a figure which shows the relationship between the density | concentration of a cationic polymer, and the signal intensity ratio obtained from an in-stem beacon type probe. It is a figure which shows the relationship between the target nucleic acid density | concentration and the signal intensity ratio obtained from an in stem beacon type probe in presence and absence of a cationic polymer.

  The present invention relates to a hybridization agent for an in-stem beacon type probe and use thereof. The cationic polymer disclosed in the present specification promotes quenching of the beacon-type probe in the absence of the target nucleic acid, and promotes hybridization with the target nucleic acid in the presence of the target nucleic acid to achieve hybridization equilibrium. Can be moved to the hybridization side with the target nucleic acid. In other words, the cationic polymer increases to an equilibrium state that is favorable for improving the detection sensitivity of the in-stem beacon probe, depending on the presence / absence of the target nucleic acid relative to the in-stem beacon probe and the state of the stem chain. Hybridization can be promoted and stabilized. Therefore, according to the present hybridization agent, it is possible to provide an improvement in detection sensitivity more than intended in the in-stem beacon type probe while ensuring hybridization ability. Further, since the hybridization state with the target nucleic acid is stabilized, the reproducibility of hybridization and the detection accuracy of hybridization are improved.

  In particular, when there are relatively few pairs of fluorescent dyes and quenching dyes introduced into the stem duplex, for example, when there is only one, the cationic polymer promotes and stabilizes quenching in the absence of the target nucleic acid. This contributes to an improvement in detection sensitivity. In addition, when there are a relatively large number of fluorescent dyes and quenching dyes introduced into the stem duplex, for example, when there are four or more pairs, the cationic polymer is doubled with the target nucleic acid in the presence of the target nucleic acid. By promoting and stabilizing chain formation, it is possible to contribute by improving detection sensitivity. Further, when the number of pairs is intermediate between them, the detection sensitivity is improved by both actions. As a result, the detection sensitivity can be improved with any type of in-stem beacon type probe.

  Hereinafter, various embodiments disclosed in this specification will be described in detail.

(In-stem beacon type probe hybridization agent)
The hybridization agent disclosed in the present specification is used for hybridization with a target nucleic acid of an in-stem beacon type probe. The in-stem beacon type probe is a beacon type probe having a stem region (stem duplex) and a loop region. A beacon type probe consists of a single-stranded oligonucleotide, the stem region is a region that has complementary base sequences that can form a duplex (stem duplex), and the loop region recognizes the target nucleic acid. It has a base sequence for The oligonucleotide constituting the beacon probe is not limited to the backbone of DNA, RNA, DNA / RNA chimera, PNA, etc., but at least the stem region and the loop region have natural nucleobases (A, T, C, G, U and the like) and a base that forms a base pair. The in-stem beacon type probe is not particularly limited as long as it has a stem region and a loop region. Thus, the in-stem beacon probe may be capable of forming multiple stem duplexes and multiple loop regions, such as tRNA or similar structures. In addition, the stem duplex in the in-stem beacon type probe is not limited to one configured on both ends of the oligonucleotide chain. One stem region may be at a portion other than the probe end and the other stem region may be at the probe end, or both stem regions may be at portions other than the probe end.

  The beacon probe disclosed in the present specification has at least one fluorescent dye and at least one quenching dye in the stem region, and these two elements are all alkylene having 2 or 3 carbon atoms. It can be linked to the oligonucleotide by a chain-phosphate backbone. Such an in-stem beacon type probe is disclosed in Patent Document 1 (WO2010 / 001902 pamphlet). Such an alkylene acid-phosphate backbone is represented by, for example, the following formulas (1) and (2).

  The length of the stem region that forms the stem duplex is not particularly limited, but each stem region can have a chain length that forms 4 to 9 base pairs. The beacon type probe preferably has a chain length that forms 8 to 40 base pairs, more preferably 15 to 30.

  Moreover, the length of the loop chain capable of forming a loop is not particularly limited, but may be about 5 or more and 40 or less. For example, the molecular beacon is preferably 8 or more and 30 or less, more preferably 10 or more and 20 or less.

  In the in-stem beacon type probe, the fluorescent dye and the quenching dye may be provided in the backbone of the stem duplex. That is, it may be provided in the backbone constituting the end such as the 5 'end or the 3' end, or may be provided inside the backbone. From the viewpoint of quenching property, the fluorescent dye and the quenching dye are preferably provided inside the terminal.

  At least one fluorescent dye and one quenching dye are provided in one stem duplex. The pair of fluorescent dyes and quenching dyes is not limited to one each, as long as it emits light when it is quenched and hybridized with the target nucleic acid. Considering hybridization with nucleic acids, each of the fluorescent dye and the quenching dye is more than a pair of three or more sets in which one or two or more quenching dyes are paired with two consecutive fluorescent dyes. Preferably, one set of pairs is formed.

  When a plurality of fluorescent dyes and quenching dyes are provided in one stem duplex, different types of fluorescent dyes may be used, or the same fluorescent dye may be used.

  The fluorescent dye is disposed in the stem duplex (stem region) as described above, but is preferably disposed in the probe sequence of the beacon type probe. This is because when a fluorescent dye forms a double strand by hybridization with a target nucleic acid depending on its type, fluorescence may be enhanced by intercalation within the double strand. Such a fluorescent dye will be described later.

  The fluorescent dye is contained as X in the unit represented by the formula (1) between the nucleotides in the stem region.

The fluorescent dye (X) is connected to a unit portion other than Y as a part of the unit, and is consequently provided in the stem. In the formula (1), R1 means an alkylene chain which has 2 or 3 carbon atoms and may be substituted. R2 means an alkylene chain which has 0 to 2 carbon atoms and may be substituted. However, R2 is preferably bonded to the second carbon atom from the oxygen atom on the 5 'side of the polyalkylene chain of R1. Z may be a direct bond or a linking group with a fluorescent dye and is not particularly limited. For example, -NHCO-, NHCS-, CONH-, -O- etc. or those containing these groups can be mentioned.

  The substituents for R1 to R2 are unsubstituted or substituted with a halogen atom, hydroxyl group, amino group, nitro group, or carboxy group by a person skilled in the art, preferably 1 to 10, and more preferably 1 Alkyl group or alkoxy group of ˜4; carbon atom of 2-20, preferably 2-10, more preferably 2-4, unsubstituted or substituted with halogen atom, hydroxyl group, amino group, nitro group, carboxy group, etc. Group or alkynyl group; a hydroxyl group, a halogen atom, an amino group, a nitro group, a carboxy group, and the like. Furthermore, a hydroxyl group, a halogen atom, an amino group, a nitro group or a carboxy group can be mentioned. The substituent of R1 may be linked to any carbon atom of the alkylene chain, but is preferably linked to the second or third carbon atom from the 5 'oxygen as in R2.

  Regarding the fluorescent dye and the quenching dye paired in the stem, the carbon number of the alkylene chain in R1 in the formulas (1) and (2) may be the same or different.

  For example, the following are mentioned as a unit represented by Formula (1) or Formula (2).

  In the formula (1), X represents a fluorescent dye. Examples of the fluorescent dye include cyanine dyes, merocyanine dyes, acridine dyes, coumarin dyes, ethidium dyes, flavin dyes, condensed aromatic ring dyes, xanthene dyes, and the like. . Of these, cyanine dyes, coumarin dyes, ethidium dyes, condensed aromatic ring dyes, and xanthene dyes can be preferably used. More preferably, it is any selected from the group consisting of Cy3, Cy5, thiazole orange, oxazole yellow, perylene, fluorescein, rhodamine, tetramethylrhodamine and Texas red. Among these, it is preferable to use a cyanine dye such as thiazole orange. The compound used as the fluorescent dye may be appropriately derivatized in order to be connected to the unit represented by the formula (1), or between the linking group in consideration of the pairing with the quenching dye, An optionally substituted alkylene chain, alkenylene group, or alkynylene group may be added. Moreover, the atom on the fluorescent dye connected with respect to the unit represented by Formula (1) is not specifically limited.

  Examples of fluorescent dyes whose fluorescence is enhanced by intercalation include thiazole orange, oxazole orange, and perylene. These fluorescent dyes are preferably disposed inside the probe array and intercalated. On the other hand, if the fluorescence intensity of the fluorescent dye is reduced by intercalation due to the combination of the fluorescent dye and R1 and R2, the beacon so that the fluorescent dye is not intercalated and the quenching dye is intercalated. It is preferable to design.

  Examples of the fluorescent dye (X) linked in Formula (1) include the following. In addition, the various compounds of the following examples are illustrations, and all the hydrogen atoms may be appropriately substituted with a substituent. In addition, an alkylene group, alkenylene group, or alkynylene group which may be substituted may be interposed in a connecting portion (dotted line portion) to the connecting group.

  The quenching dye is contained in the unit represented by the following formula (2) between the nucleotides of the stem-forming strand. That is, the quenching dye (Y) is connected to a unit part other than Y as a part of the unit, and as a result, provided in the stem.

  In the formula (2), R1, R2 and Z have the same meanings as in the formula (1). Moreover, the substituents of R1 to R2 are also synonymous with those in the formula (1).

  In formula (2), Y represents a quenching dye. The quenching dye is not particularly limited as long as the fluorescent dye to be used can be quenched by forming the stack structure disclosed in this specification, and examples thereof include azobenzene and derivatives thereof. These are all photoisomerizable compounds, but are thought to exhibit a quenching action due to the presence of -N = N-. The quenching dye is preferably any one selected from methyl red, azobenzene, and methylthioazobenzene. A typical azo quenching dye is a type of quenching dye whose fluorescent dye has a lower HOMO than that of the quenching dye in relation to a polycyclic condensed fluorophore such as perylene, in other words, a quenching dye having a strong reducing power. It can be said that there is.

  In addition, examples of the quenching dye include compounds having an anthraquinone skeleton. As the compound having an anthraquinone skeleton, in addition to anthraquinone, one or two or more hydrogen atoms bonded to the carbon atom constituting the benzene ring of anthraquinone may be a hydroxyl group, a nitro group, an amino group, a sulfonic acid group, a halogen atom, Examples include various anthraquinone derivatives substituted with one or more functional groups selected from an alkylamino group and the like. Various anthraquinone derivatives can be easily obtained from, for example, Tokyo Chemical Industry Co., Ltd. For example, natural examples of the anthraquinone derivative include compounds of cochineal dyes, red dyes such as alizarin, and rack dyes. As an anthraquinone derivative, any anthraquinone derivative having a LUMO lower than that of a fluorescent dye can be used. However, an electron-withdrawing substitution that does not increase the LUMO energy level, such as a nitro group or a carboxyl group. Groups are preferred. Anthraquinone is preferred. Typical anthraquinone-based quenching dyes such as anthraquinone are quenching dyes that satisfy the condition that the LUMO of the fluorescent dye is higher than the LUMO of the quenching dye in relation to the fluorophore of the condensed aromatic ring system such as perylene. It can be called a quenching dye.

  The quenching dye preferably has a maximum absorption wavelength of 150 nm or less from the maximum absorption wavelength of the fluorescent dye forming a pair, from the viewpoint of the quenching ability. The difference between the maximum absorption wavelength of the quenching dye and the maximum absorption wavelength of the fluorophore is more preferably within 100 nm, and even more preferably within 50 nm. Furthermore, it is preferable that the maximum absorption wavelength of the quenching dye is on the shorter wavelength side than the maximum absorption wavelength of the fluorescent dye. The quenching dye can be optimized by controlling the maximum absorption wavelength of the quenching dye in relation to the fluorescent dye used.

  Control of the maximum absorption wavelength of the quenching dye (shift to the long wavelength side or the short wavelength side) is possible, for example, by introducing an electron-withdrawing group or an electron-donating group into the basic skeleton of the quenching dye. . For example, as shown in the following [Chemical 8B], an electron-withdrawing group (for example, nitro group, halogen) or electron-donating group (for example, nitro group or halogen) is located in the ortho or para position with respect to the azo group of the azobenzene-based skeleton such as methyl red For example, it is conceivable to introduce a methoxy group) as appropriate.

  The compound used as the quenching dye may be appropriately derivatized in order to be linked to the unit represented by the formula (2), or substituted between the linking groups in consideration of pairing with the quenching dye. An alkylene chain which may be used may be added. Moreover, a connection part with the connection group of a quenching dye is suitably selected.

  Examples of the quenching dye (Y) linked in the formula (2) include the following. In addition, the various compounds of the following examples are illustrations, and all the hydrogen atoms may be appropriately substituted with a substituent. In addition, an alkylene group, alkenylene group or alkynylene group which may be substituted may be interposed in a connecting portion (dotted line portion) to the connecting group. Moreover, the atom on the fluorescent dye connected with respect to the unit represented by Formula (2) is not specifically limited.

  Introducing fluorescent dyes and quenching dyes can be achieved, for example, by using an amidite derivative that introduces this type of unit in place of the amidite derivative corresponding to the nucleotide in a normal solid phase synthesis method. For example, the amino group of aminoalkyldiols such as D-threonyl and 3-amino1,2-propanediyl is protected with an appropriate protecting group such as allyloxycarbonyl group, and one hydroxyl group is protected with dimethoxytrityl chloride or the like. Then, 2-cyanoethyl N, N, N, N-tetraisopropyl phosphorodiamidide is introduced into the other hydroxyl group. Then, a fluorescent dye or a quenching dye may be introduced into this amidite body to form an amidite monomer. For example, with respect to azobenzene compounds and perylene compounds, for example, the method described in Nature Protocols, Vol. 2, pages 203 to 212, Journal of the American Chemical Society can be used. (Journal of the American Chemical Society) 2003, 125, 2217-2223, and the method described in Tetrahedron Letters, 2007, 48, 6759-6762 can be applied. . When such a monomer is obtained, a fluorescent dye or a fluorescent dye can be added to a desired site according to a conventionally known DNA synthesis method, for example, the method described in Nature Protocols 2007, Vol. 2, pages 203 to 212. Oligonucleotides comprising units linked to quenching dyes can be synthesized.

  In addition, the fluorescent dye or the quenching dye may be introduced after synthesis of an oligonucleotide having the above-mentioned amidite body in which the amino group is protected with an allyloxycarbonyl group or the like at a desired position. For example, after deprotecting an amino group from an oligonucleotide having a unit in which the amino group is protected on a CPG carrier, a fluorescent dye that introduces or holds a carboxylic acid group or an isocyanate group that can react with the amino group A fluorescent dye or the like may be introduced by reacting with a quenching dye.

  The in-stem beacon type probe disclosed in the present specification described above is a probe that self-generates a fluorescent signal, so that it is not necessary to label the target nucleic acid with a fluorescent substance or the like, and the target nucleic acid can be freely selected. There is an advantage that detection can be performed with high specificity and high specificity. Furthermore, since the fluorescent dye and the quenching dye are provided in the stem duplex, it is possible to avoid or suppress the influence of the immobilization on the light emission / quenching as well as the ease of immobilization on the solid phase carrier.

  The in-stem beacon type probe disclosed in this specification is used to detect target nucleic acids in a liquid phase such as real-time PCR to which a conventional molecular beacon is applied, as well as analysis of gene expression patterns such as gene expression patterns, disease classifications, and diseases. It is advantageous for gene expression monitoring such as gene diagnosis, polymorphism detection such as SNP, and detection of various mutations. In particular, detection of polymorphisms and mutations, more comprehensive detection of polymorphisms and mutations, and detection of a plurality of target nucleic acids having multiple SNPs and mutation sites, preferably several dozens or more, simultaneously with high sensitivity and high accuracy Will be able to.

  The in-stem beacon type probe is used in various forms. For example, it may be in the form of a solution for hybridization in a liquid phase, or may be in a form that dissolves in a suitable medium at the time of use. Alternatively, it may be immobilized on a suitable solid phase carrier for solid phase-liquid phase hybridization. A solid phase carrier for immobilizing a probe composed of an oligonucleotide or the like and a method for immobilizing the probe on the solid phase carrier are technical common knowledge of those skilled in the art at the time of filing the present application. By selecting a solid phase carrier or selecting an immobilization technique, a solid phase carrier on which an in-stem beacon type probe is immobilized can be obtained.

(Target nucleic acid)
The target nucleic acid is a nucleic acid that is recognized by the in-stem beacon type probe disclosed in the present specification and is subject to hybridization. Here, the nucleic acid may include DNA, RNA, a nucleic acid derivative, a non-natural nucleotide of a modified base, or a non-natural nucleotide bond called PNA (Nature Biotechnology, 14.1700-1704 (1996)). The nucleic acid may be a single-stranded nucleic acid, a double-stranded nucleic acid, or a triple-stranded nucleic acid, unless otherwise specified. Double-stranded nucleic acids are complementary base pairs, such as adenine (A) and thymine (T) and guanine (G) and cytosine (C) in DNA, and A and uracil (U) and G in RNA. This means a higher order structure formed by pairing with C. In addition to DNA single-stranded / DNA single-stranded double-stranded DNA, RNA single-stranded / RNA single-stranded double-stranded RNA, DNA single-stranded / RNA single-stranded hybrid double-stranded nucleic acid may also be mentioned.

  The target nucleic acid includes target sequences such as nucleotide polymorphisms and mutations corresponding to the intention of hybridization. Polymorphisms and mutations include substitution or deletion of one or more nucleobases. Upon hybridization with the in-stem beacon type probe, the target nucleic acid can be in a solution state or the like.

(Cationic polymer)
The cationic polymer refers to a polymer having a polymer chain having a cationic group. Such a cationic polymer may have a polymer chain (part) having a cationic group. Examples of the cationic polymer chain include polylysine and polyallylamine. The cationic polymer may be a copolymer in which a hydrophilic polymer chain such as sugar is grafted as a branched chain on the cationic polymer chain. Examples of such hydrophilic polymer chains include dextran and polyethylene glycol.

  Various cationic polymers have already been reported. For example, Japanese Laid-Open Patent Publication No. 2001-78769 and Bioconjugate Chem., 8, 3-6 (1997) disclose cationic polymer polymers for promoting exchange between nucleotide sequences. This is a polymer obtained by graft polymerization using a cationic polymer such as polylysine or polyarginine as a main chain and dextran or polyethylene glycol as a side chain. Examples thereof include α-PLL-g-Dex, ε-PLL-g-Dex, and PAA-g-Dex.

Dextran side chain modified α-poly (L-lysine) (hereinafter abbreviated as α-PLL-g-Dex)

Dextran side chain modified ε-poly (L-lysine) (hereinafter abbreviated as ε-PLL-g-Dex)

  Dextran side chain modified polyallylamine (hereinafter abbreviated as PAA-g-Dex)

  The hybridization agent may be used together with the in-stem beacon type probe in hybridization using the in-stem beacon type probe, or may be provided separately from the in-stem beacon type probe. On the other hand, it may be in the form of containing the hybridization agent and the in-stem beacon type probe at the time of use or in a dilutable concentration at the same time in an appropriate medium. Moreover, the form which melt | dissolves in a suitable medium at the time of use may be sufficient.

(Hybridization kit)
The hybridization kit disclosed in this specification includes a hybridization agent and an in-stem beacon type probe. The in-stem beacon type probe in the hybridization kit takes a form prepared separately from the hybridization agent. The in-stem beacon type probe is preferably included in the hybridization kit in a form in which different ones are immobilized on a solid phase carrier. The use of hybridization agents improves the detection sensitivity of in-stem beacon probes immobilized on a solid phase carrier, and the fluorescence quenching and emission levels of the fluorescent dye are homogenized to reproduce hybridization on the solid phase carrier. And detection accuracy can be greatly improved.

  As described above, various types of solid-phase carriers and immobilization forms of in-stem beacon probes can be used. Those skilled in the art appropriately use known materials and known methods as needed. By applying, a solid phase carrier on which an in-stem beacon type probe is immobilized can be obtained.

(Hybridization method)
The hybridization method disclosed in the present specification is a method for hybridizing an in-stem beacon type probe and a target nucleic acid, in the presence of a hybridization agent, the target nucleic acid and the in-stem beacon type probe. A step of obtaining a signal based on the fluorescent dye of the in-stem beacon type probe in the presence of the target nucleic acid (hereinafter also referred to as a first step) by contacting under conditions where hybridization is possible. it can. According to such a process, the duplex formation between the target nucleic acid and the in-stem beacon probe can be promoted and stabilized, so that the fluorescent dye can emit light with high intensity. Further, the light can be emitted uniformly with good reproducibility.

  The hybridization method further includes a step of obtaining a signal based on the fluorescent dye of the in-stem beacon type probe in the presence of the hybridization agent in the absence of the target nucleic acid (hereinafter also referred to as the second step). Can be provided. According to such a process, it is possible to promote and stabilize the formation of the stem duplex of the in-stem beacon type probe and effectively quench it. In addition, it can be extinguished uniformly with good reproducibility.

  By including either the first step or the second step, the detection sensitivity of the in-stem beacon probe can be improved. Further, by providing these two steps, the detection sensitivity of any in-stem beacon type probe can be improved.

  The in-stem beacon type probe used for the hybridization method preferably has two or three combinations of one fluorescent dye and one quenching dye in the stem region. In the case of such a stem region, the detection sensitivity is improved both by promoting and stabilizing stem duplex formation and by promoting and stabilizing duplex formation with the target nucleic acid. It is also preferable that four or more in-stem beacon probes have the combination in the stem region. With such an in-stem beacon type probe, duplex formation with the target nucleic acid is promoted and stabilized, and the emission intensity of the fluorescent dye can be greatly enhanced.

  The hybridization conditions between the in-stem beacon type probe and the target nucleic acid in the hybridization method are not particularly limited, and the known hybridization conditions such as temperature, pH, solvent, time, stirring mode, etc. What is necessary is just to select suitably.

EXAMPLES Hereinafter, although an Example is given and this invention is demonstrated concretely, this invention is not limited to a following example.

  Poly (L-lysine) -graft-dextran (PLL-g-Dex) as a cationic polymer was synthesized according to Japanese Patent Publication No. 2001-78769 and Bioconjugate Chem., 8, 3-6 (1997). PLL-g-Dex having a molecular weight of L-lysine) of 8,000 and a dextran graft ratio of 90% by weight was obtained.

Synthesis of DNA incorporating perylene and Cy3 as fluorescent dyes and anthraquinone and nitromethyl red as corresponding quenching dyes is described in Tetrahedron Lett., 2007, 48, 6759-6762 (perylene), Chem. Commun., 2006, 5062. -5064 (anthraquinone), Chem. Eur. J., 2009, 15, 10092-10102 (nitromethyl red), Angew. Chem. Int. Ed., 2010, 49, 5502-5506 (Cy3), First, phosphoramidite monomers corresponding to these were synthesized, and synthesized and purified by using the methods described in Nature Protocols 2007, Vol. 2, pages 203 to 212. Perylene, Cy3, nitromethyl red, and anthraquinone used for phosphoramidite monomer synthesis are shown below. The C3 alkylene chain-phosphate backbone and cationic polymer having these fluorescent dyes and quenching dyes are also shown below.

  The base sequence of the synthesized DNA is shown below.

MB-EQ-t: 5'- ETGGTC -CTTGAGAAAGGGC- GACCAQ -3 '
MB-EQ-1 ： 5'- TGGETC -CTTGAGAAAGGGC- GAQCCA -3 '
MB- EQ-2: 5'- TEGGETC -CTTGAGAAAGGGC- GAQCCQA -3 '
MB- EQ-3: 5'- GEGTEGGETC -CTTGAGAAAGGGC- GAQCCQACQC -3 '
MB- EQ-4: 5'- GECGEGTEGGETC -CTTGAGAAAGGGC- GAQCCQACQCGQC -3 '
MB-YN: 5'- TGYGTC CTTGAGAAAGGGC GACNCA -3 '
(The underlined part constitutes the stem part)
Surv-1: 3'-ACGCCACCAGGAACTCTTTCCCG-5 '(target)
Surv-2: 3'-GAACTCTTTCCCGCTGGT-5 '(target)

  Concentrated beacon, target, and polymer are dissolved in 200 μl phosphate buffer, transferred to a cell, set in a fluorometer, allowed to stand at 80 ° C. for 5 minutes, and then 80 ° C. to 20 ° C. at 5 ° C. / The temperature was decreased at min and the fluorescence spectrum was measured after standing for 5 minutes, thereby measuring the fluorescence intensity in the presence of the target nucleic acid and polymer in the equilibrium state in the presence of the target nucleic acid. Moreover, the fluorescence intensity in the state where the beacon was closed in the equilibrium state in the absence of the target nucleic acid was estimated by measuring the fluorescence spectrum under exactly the same conditions as above except that no target was added.

In addition, as a comparative example, by using only a beacon and a target, the fluorescence spectrum is measured under the same conditions as above to estimate the fluorescence intensity without a polymer, and further by measuring the fluorescence spectrum under the same conditions as above only with a beacon The fluorescence intensity with the beacon closed was estimated. The fluorescence spectrum was measured using a fluorimeter with temperature controller FP-6500 manufactured by JASCO Corporation under the following conditions.
Excitation side bandwidth: 5 nm Fluorescence side bandwidth: 5 nm
Response: 0.1sec Sensitivity: Medium
Measurement range: 435-650nm Excitation wavelength: 425nm
Temperature: 20 ° C

  The results are shown below. In the tables below, all beacon probes were set to 0.2 μM. The phosphate buffer pH 7.0 was prepared as a 100 mM NaCl 10 mM sodium phosphate aqueous solution. The amount of the added cationic polymer was such that N / P = 2 when [Surv-1] / [MB-EQ-t] = 1. The N / P ratio mentioned here means (amount of amino group of cationic polymer) / (amount of phosphate group of DNA). S / N refers to the ratio of the fluorescence intensity in the presence of the target nucleic acid and in the absence of the target nucleic acid.

  As shown in the above table, an improvement in the S / N ratio was observed by adding cationic polymer in all systems. In particular, when a cationic polymer is added, the fluorescence intensity decreases due to stabilization of the stem site in the absence of the target nucleic acid (background noise decreases), and stabilization of duplex formation with the beacon in the presence of the target nucleic acid. Increased fluorescence intensity (signal enhancement) was observed as a result of which S / N ratio was improved. In particular, in MB-EQ-3 (having three pairs of fluorescent dye and quenching dye in the stem region), the effect of adding a cationic polymer was remarkable, and the S / N ratio increased to about 600 times.

In this example, the response speed of the beacon synthesized in Example 1 was measured by the following procedure.
(1) Prepare a beacon and a target in separate tubes (prepared so as to have an appropriate concentration when dissolved in 2000 μl of buffer).
(2) Beacon (+ polymer) 1980 μl of phosphate buffer (same composition as Example 2)
(3) Dissolve the target in 20 μl of buffer (combined with (2) to make 2000 μl).
(4) Transfer the buffer solution of the beacon to the cell, turn the stirring bar, and leave it for 5 minutes (20 ° C.).
(5) Start measurement after 5 minutes, and add the target after 5 minutes from the start.
(6) Continue the measurement for 2 hours.

(Measurement conditions for MB-EQ system)
Excitation side bandwidth: 3 nm Fluorescence side bandwidth: 3 nm
Response: 0.1sec Sensitivity: Medium
Measurement range: 0 to 7200 sec Data acquisition interval: 1 sec
Excitation wavelength: 425 nm Fluorescence wavelength: 460 nm
Temperature: 20 ° C

(Measurement conditions for MB-YN)
Excitation side bandwidth: 3 nm Fluorescence side bandwidth: 3 nm
Response: 0.2 sec Sensitivity: Medium
Measurement range: 0 to 7200 sec Data acquisition interval: 1 sec
Fluorescence wavelength: 564 nm Excitation wavelength: 546 nm
Temperature: 20 ° C

  The measurement results for the MB-EQ system are shown in the following table. The beacon type probe was 0.2 μM, and [Surv-1] = 0.4 μM. The amount of the added cationic polymer was N / P = 2 when [Surv-1] / [MB-EQ] = 1.

  As shown in Table 6, the response speed was significantly improved by adding the cationic polymer.

  Moreover, the measurement result about MB-YN is shown in FIG. In FIG. 1, [MB-YN] = 0.2 μM and [Surv-2] = 0.4 μM were fixed, and the cationic high molecular weight was N / P = 0 (comparative example), 0.2 (invention), 0.5 ( The present invention), 1 (present invention), and 2 (present invention) were measured.

  As shown in FIG. 1, the response speed was remarkably increased by increasing the addition amount of the cationic polymer.

  From the above, it was found that the cationic polymer is a hybridization agent that can be widely and powerfully applied to improve the detection sensitivity of the in-stem beacon type probe. It was also found that the effect can be enhanced by increasing the supply amount within a certain range of the supply amount.

  In this example, the stability of the fluorescence intensity based on the fluorescent dye of the probe was confirmed when the in-stem beacon type probe was immobilized on a plastic substrate and hybridized in the presence and absence of the target nucleic acid. . The signal intensity ratio was also confirmed. Two types of beacon probes were prepared according to the sequences of two types of target nucleic acids.

  As a substrate, a plastic substrate manufactured by Sumitomo Bakelite Co., Ltd. was used, and b3a2-T or b2a2-T was used as a target nucleic acid at a concentration of 0 to 10 nM. For hybridization, 1 × SSC (NaCl: 150 mM) + 0.5% SDS or 1 × SSC was used. Hybridization was carried out in a stationary state, the reaction temperature being 65 ° C. or 45 ° C., the reaction time being 16 hours, and sealing with glass. In addition, hybridization was performed both in the presence (200 μM or 2-200 μM) and absence of the cationic polymer synthesized in Example 1.

The fluorescent dye and the quenching dye in the beacon type probe were Cy3 and nitromethyl red (N). The beacon type probe and the target nucleic acid had the following sequences, and the beacon type probe was synthesized according to Example 2.
(Beacon probe array)
b2a2-A5-dRY2: 5'-X TTTTT- TGANNAG GGCTTCTTCCTTAT CTYTCA -3 '
b3a2-A5-dRY2: 5'-XTTTTT- TGANNAG GGCTTTTGAACTCTG CTYTCA -3 '
(N = nitromethyl red, Y = Cy3, X is represented by the following formula. The underlined part constitutes the stem part)

(Target nucleic acid sequence)
b2a2: 5'-ATAAGGAAGAAGCCCTTCA-3 '
b3a2: 5'-CAGAGTTCAAAAGCCCTTCA-3 '

  Using b3a2-A5-dRY2 as a match probe and b2a2-A5-dRY2 as a mismatch probe, it is spotted on the substrate in the form shown in FIG. 2, and the target nucleic acid b3a2- is present in the presence (200 μM) and absence of a cationic polymer. The result of hybridization with T (100 nM) and detection of fluorescence is shown in FIG. As shown in FIG. 3, by the presence of the cationic polymer, the fluorescence of the match probe was enhanced and its level became uniform, the mismatch probe was well quenched and its level became uniform.

  Further, the fluorescence signal intensity ratio obtained from the hybridization of the match probe b3a2-A5-dRY2 and the target nucleic acid b3a2-T (10 nM) when the concentration of the cationic polymer is changed (0, 2, 20, and 200 μM). The measurement results are shown in FIG. As shown in FIG. 4, when the concentration of the cationic polymer was increased, the signal intensity ratio obtained from the match probe was also improved accordingly.

  Further, when the target nucleic acid b2a2 (match) and b3a2 (mismatch) are supplied (0 to 10 nM) to the match probe (b2a2-A5-dRY2) for the target nucleic acid b2a2, in the presence of a cationic polymer (200 μM) and non- FIG. 5 shows the measurement results of the target nucleic acid concentration in the presence and the signal intensity ratio obtained from the b2a2 match probe. As shown in FIG. 5, in the presence of a cationic polymer, the signal intensity ratio obtained from the match probe increases with the change in the concentration of the target nucleic acid b2a2, while the concentration change in the target nucleic acid b3a2 that is a mismatch. It was found that a low signal intensity ratio can be stably maintained and measurement with good detection sensitivity, accuracy and reproducibility is possible. In contrast, in the absence of a cationic polymer, the signal intensity ratio obtained from the match probe increases according to the concentration of the target nucleic acid b2a2, but is unstable and compared with the target nucleic acid b3a2 that is a mismatch. High signal intensity ratio. From the above results, it was found that in the presence of a cationic polymer, hybridization with good detection sensitivity, reproducibility and accuracy is possible even when the target nucleic acid concentration is as low as 10 nM or less. .

Claims (15)

Cationic polymer as an active ingredient,
A hybridization agent for an in-stem beacon type probe, which is a beacon type probe having a loop region and a stem region, and having a fluorescent dye and a quenching dye in the stem region. The hybridization agent according to claim 1, wherein each of the fluorescent dye and the quenching dye is provided in the stem region of the in-stem beacon type probe as the following units.
(In the formula, X represents a fluorescent dye, R 1 represents an alkylene chain having 2 or 3 carbon atoms and may be substituted, and R 2 has 0 to 2 carbon atoms and is substituted. Represents an alkylene chain which may be substituted, and Z represents a direct bond or a linking group.)
(In the formula, Y represents a quenching dye, R 1 represents an alkylene chain having 2 or 3 carbon atoms, which may be substituted, and R 2 has 0 to 2 carbon atoms and is substituted. Represents an alkylene chain which may be substituted, and Z represents a direct bond or a linking group.)   The hybridization agent according to claim 1 or 2, wherein the in-stem beacon type probe has two or three combinations of one each of the fluorescent dye and the quenching dye in the stem region.   The hybridization agent according to claim 3, wherein the in-stem beacon type probe has four or more combinations in the stem region.   The hybridization agent according to claim 1, wherein the cationic polymer is a copolymer composed of a polymer chain having a cationic group and a hydrophilic polymer chain.   The hybridization agent according to claim 5, wherein the polymer chain having a cationic group is polylysine or polyallylamine.   The hybridization agent according to claim 5 or 6, wherein the hydrophilic polymer chain is dextran or polyethylene glycol.   The hybridization agent according to any one of claims 1 to 7, wherein the cationic polymer contains at least one of α-PLL-g-Dex, ε-PLL-g-Dex, and PAA-g-Dex.   The hybridization agent according to any one of claims 1 to 8, further comprising the in-stem beacon type probe. The hybridization agent according to any one of claims 1 to 8,
A beacon type probe having a loop region and a stem region, the in-stem beacon type probe having a fluorescent dye and a quenching dye in the stem region;
A hybridization kit comprising:   The hybridization kit according to claim 10, wherein the in-stem beacon type probe has one or more in-stem beacon type probes immobilized on a solid phase carrier. A hybridization method between an in-stem beacon type probe and a target nucleic acid,
In the presence of the hybridization agent according to any one of claims 1 to 8, the target nucleic acid, a loop region and a stem region, and a fluorescent dye and a quenching dye are contained in the stem region. A method of obtaining a signal based on the fluorescent dye of the in-stem beacon probe in the presence of the target nucleic acid by contacting the stem beacon probe with a hybridizable condition.   The method according to claim 12, further comprising obtaining a signal based on the fluorescent dye of the in-stem beacon probe in the presence of the hybridization agent in the absence of the target nucleic acid.   The method according to claim 12 or 13, wherein the in-stem beacon type probe has two or three combinations of the fluorescent dye and the quenching dye in the stem region. The method according to claim 12 or 13, wherein the in-stem beacon type probe has four or more combinations in the stem region.