JP5920763B2 - Fluorescently labeled oligonucleotide derivatives and uses thereof - Google Patents

Description

  The present invention relates to fluorescently labeled oligonucleotide derivatives and uses thereof.

  Hybridization based on the bases of nucleic acids forming base pairs with complementary bases is used in a wide range of applications such as identification, detection, and quantification of target nucleic acids having a target base sequence (target sequence). Yes. The target base sequence forms a duplex by hybridization with a probe having a complementary base sequence. Several approaches have been attempted as probes for specifically detecting such duplexes.

  One is a molecular beacon. A molecular beacon is composed of a single-stranded DNA loop complementary to a target sequence and a double-stranded stem annealed by a complementary strand arm designed to sandwich the probe. The end of the stem is equipped with a fluorescent dye and a quencher that quenches the fluorescent dye. At the time of stem formation, the fluorescent dye is quenched by the quencher, but when the target nucleic acid and the probe are hybridized, the stem is unwound. Thus, the fluorescent dye and the quencher are separated, the fluorescent dye emits light, and the hybridized duplex can be detected.

  As such molecular beacons, fluorescent dyes are arranged at both ends of the stem so that they associate and quench, and the associated state is released by hybridization with the target nucleic acid to emit the original fluorescence (Patent Literature). 1). DNA in which fluorescent dyes arranged on a plurality of probes on a DNA probe array are associated with fluorescent dyes of other adjacent probes, and further, probes that use quenching due to self-association of fluorescent dyes and light emission due to their elimination are immobilized. A probe array has also been proposed (Patent Document 2).

  Furthermore, by introducing two dyes such as thiazole orange through a single-stranded thymine such as DNA, a system in which fluorescence derived from the dye emits light only when a complementary base sequence is hybridized has been studied ( Patent Document 3).

JP 2005-80637 A JP 2002-281978 A JP 2009-171935 A

  However, in the molecular beacon disclosed in Patent Document 1 and the like, a stem structure for quenching a fluorescent dye is essential, but it is necessary to disrupt this stem structure for hybridization with a target nucleic acid. There is a problem that response is slow. In addition, the method described in Patent Document 2 has a problem of accuracy and sensitivity in quenching due to self-association between fluorescent dyes provided in adjacent probes. Furthermore, the method described in Patent Document 3 has a problem that the extinction state is not always sufficient and the synthesis cannot be performed efficiently.

  Therefore, in the present invention, in order to detect hybridization of a target nucleic acid, a fluorescently labeled oligonucleotide derivative capable of effectively realizing a quenching state when non-hybridized with a target nucleic acid and a light-emitting state when hybridizing and the use thereof are provided. One purpose is to provide.

  The present inventors have made various studies on effectively forming a quenching state without using a stem chain and a quencher. As a result, by introducing a monomer unit having a fluorescent dye in a predetermined arrangement state in the polymer chain of the nucleotide derivative, an effective quenching state can be formed with a simple structure at the time of non-hybridization, and at the same time, fluorescence at the time of hybridization. It was found that the dye emits light. Based on these findings, the following means are provided.

（式中、Ｘは、蛍光色素を表し、Ｒ1は、炭素数が２又は３であって置換されていてもよいアルキレン鎖を表す。また、Ｒ2は、炭素数が０以上２以下であって置換されていてもよいアルキレン鎖を表し、Ｚは、直接の結合又は連結基を表す。Ａ１は、水素原子、リン酸基、ホスホジエステル結合を介して連結されるヌクレオチド誘導体又はオリゴヌクレオチド誘導体を表し、Ｂ１は、水素原子、リン酸基、ホスホジエステル結合を介して連結されるヌクレオチド誘導体又はオリゴヌクレオチド誘導体を表す。） (1) a fluorescently labeled oligonucleotide derivative,
Comprising a single-stranded polymer chain comprising at least a nucleotide derivative as a first monomer unit;
An oligonucleotide derivative comprising three or more second monomer units having a fluorescent dye in the polymer chain, and at least one first monomer unit between the second monomer units.
(2) The oligonucleotide derivative according to (1), wherein at least four or more of the second probes are provided in the polymer chain.
(3) The oligonucleotide derivative according to (1) or (2), wherein the number of the first monomer units arranged between the second monomer units is at least two.
(4) The oligonucleotide derivative according to any one of (1) to (3), wherein the number of the first monomer units arranged between the second monomer units is 4 or less.
(5) The oligonucleotide derivative according to any one of (1) to (4), wherein the second monomer unit is represented by the following formula (1).

(In the formula, X represents a fluorescent dye, R 1 represents an alkylene chain having 2 or 3 carbon atoms and may be substituted. R 2 has 0 to 2 carbon atoms. Represents an optionally substituted alkylene chain, Z represents a direct bond or a linking group, and A1 represents a nucleotide derivative or an oligonucleotide derivative linked via a hydrogen atom, a phosphate group, or a phosphodiester bond. , B1 represents a nucleotide derivative or an oligonucleotide derivative linked via a hydrogen atom, a phosphate group, or a phosphodiester bond.

(6) The oligonucleotide derivative according to any one of (1) to (5), comprising two or more second monomer units each having two fluorescent dyes that cause energy transfer due to resonance.
(7) The oligonucleotide derivative according to any one of (1) to (6), wherein the fluorescent dye is a condensed aromatic ring dye.
(8) a fluorescently labeled oligonucleotide derivative,
Comprising a single-stranded polymer chain comprising at least a nucleotide or a derivative thereof as a first monomer unit;
A plurality of second monomer units comprising a fluorescent dye are converted into the following (a) and (b):
(A) When non-hybridized with the target nucleic acid, they associate with each other and are self-quenched.
(B) Upon hybridization with the target nucleic acid, fluorescence is emitted by the cancellation of the association.
The oligonucleotide derivative is provided in the polymer chain so that the second monomer units are separated by the first monomer units so as to satisfy the above.
(9) An immobilized body in which the fluorescently labeled oligonucleotide derivative according to any one of (1) to (8) is immobilized on a solid phase body.
(10) A method for detecting a target nucleic acid,
A hybridization step in which the fluorescently labeled oligonucleotide derivative according to any one of (1) to (8) and a nucleic acid sample possibly containing a target nucleic acid are brought into contact with each other so as to be hybridizable;
A detection step for detecting a hybridization product of the fluorescently labeled oligonucleotide derivative and the target nucleic acid in the hybridization step under conditions where the fluorescent dye can be detected;
A detection method comprising:

It is a figure which gives an example and explains the outline of the present invention. 2 is a diagram showing a fluorescence spectrum obtained in Example 1. FIG. 2 is a diagram showing a fluorescence spectrum obtained in Example 1. FIG. 6 is a diagram showing a fluorescence spectrum obtained in Example 2. FIG. 6 is a diagram showing a fluorescence spectrum obtained in Example 3. FIG. It is a figure which shows the fluorescence spectrum obtained in Example 4.

  The disclosure of the present specification relates to a fluorescently labeled oligonucleotide derivative and use thereof, and more particularly, to a fluorescently labeled oligonucleotide derivative capable of realizing self-quenching and light emission without requiring a stem structure and a quencher. In particular, the present invention relates to a fluorescently labeled oligonucleotide derivative capable of controlling self-quenching and luminescence by hybridization with a target nucleic acid with a simple structure.

FIG. 1 shows a typical self-quenching-luminescence control form of the fluorescently labeled oligonucleotide derivative of the present invention.
According to the fluorescently labeled oligonucleotide derivative of the present invention, the polymer chain has a form in which three or more second monomer units having a fluorescent dye and at least one first monomer unit are provided between the second monomer units. . When the target nucleic acid of the fluorescence-labeled oligonucleotide derivative is absent, that is, when such a polymer chain is present in a single-stranded state, the polymer chain is in a random coil state as shown in FIG. Fluorescent dyes contained in the group associate and self-quence.

  On the other hand, when the target nucleic acid is present, when a polymer chain portion containing three or more second monomer units hybridizes with another polymer chain having a base sequence complementary to the base sequence of the portion, FIG. As shown in FIG. 3, the polymer chain forms a double chain with other polymer chains, and as a result, the fluorescent dye is intercalated into the double chain, and the mutual association state is eliminated. As a result, the fluorescent dye can emit fluorescence possessed by itself.

  As described above, according to the fluorescently labeled oligonucleotide derivative (single strand) of the present invention, the second monomer unit having a fluorescent dye can be obtained without using a quenching element such as a stem structure or a quencher. By providing three or more units separated by at least one first monomer unit, self-quenching can be performed when the target nucleic acid is absent, and light can be emitted when hybridized with the target nucleic acid.

  Furthermore, the target nucleic acid can be detected by FRET by providing at least two second monomer units each having two fluorescent dyes that can cause excitation energy transfer (FRET) by resonance.

According to such a fluorescently labeled oligonucleotide derivative, for example, the following advantages can be exhibited over the prior art described in the background of the present invention in detecting the target nucleic acid.
(1) Even if the treatment such as washing is omitted after hybridization, the target nucleic acid can be detected.
(2) Stem strands and quenchers can be eliminated.
(3) Since a self-quenching and light emission can be realized with a simple structure without requiring a complicated structure for self-quenching, the manufacturing cost of the probe can be reduced.
(4) Since it is not necessary to open the stem strand for hybridization with the target nucleic acid, highly responsive hybridization and detection are possible.
(5) Since the second monomer unit can be additionally introduced to a predetermined base sequence in the polymer chain, the fluorescent dye can be introduced at an arbitrary site and number regardless of the base sequence. Further, since the additional introduction does not inhibit the hybridization between the hybridization sequence and the target sequence, it can hybridize with the target nucleic acid with high responsiveness.
(6) The self-quenching state and the light emission state (emission intensity, etc.) can be easily adjusted by the second monomer unit number and the first monomer unit number interposed between the second monomer units.

  In this specification, “nucleic acid” refers to all DNA and RNA including cDNA, genomic DNA, synthetic DNA, mRNA, total RNA, siRNA, miRNA, hnRNA and synthetic RNA, peptide nucleic acid, morpholino nucleic acid, and locked ( Bridged) nucleic acid (LNA, BNA), SNA (Angew. Chem. Int. Ed. 2011, 50, 1285-1288), aTNA (J. Am. Chem. Soc. 2010, 132, 14702-14703), methyl phospho Artificial synthetic nucleic acids such as nate nucleic acids and S-oligonucleic acids. Moreover, it may be single-stranded or double-stranded. In the present specification, the “target nucleic acid” is an arbitrary nucleic acid having an arbitrary sequence. Typically, it may have a base sequence that serves as a genetic indicator in humans or non-human animals, such as constitution, genetic disease, onset of specific diseases such as cancer, disease diagnosis, treatment prognosis, drug and treatment selection, etc. There may be mentioned nucleic acids with Examples of the index include polymorphisms such as SNP and congenital or acquired mutations. Further, nucleic acids derived from microorganisms such as pathogenic bacteria and viruses are also included in the target nucleic acid.

  As the target nucleic acid, a sample described later or a nucleic acid fraction thereof can be used as it is. Typically, an amplification product obtained by amplifying a plurality of target nucleic acids by an amplification reaction by PCR or multiplex PCR can be used.

  In this specification, “sample” refers to a sample that may contain a target nucleic acid, and its origin and acquisition method are not particularly limited. Samples include biological cells, tissues, blood of humans and animals, urine, saliva and the like, and any sample can be used as long as it contains nucleic acids. Fractions containing nucleic acids from these various samples can be obtained by those skilled in the art with reference to conventional techniques as appropriate.

  As used herein, “target sequence” refers to a sequence consisting of one or more bases characteristic of a target nucleic acid to be detected. The target sequence may be a sequence characteristic of the target nucleic acid. The target sequence may be, for example, a partial sequence with low homology between target nucleic acids, or a sequence that is complementary or has low homology to other nucleic acids that may be contained in a sample. The target sequence may be one in which a base sequence is artificially set or a natural base sequence is changed.

  In the present specification, the “nucleotide derivative” means a compound or unit having the same quality as that of a nucleotide or a nucleotide with a specific base. Therefore, nucleotide derivatives include compounds constituting the units of various artificial nucleic acids such as PNA and LNA, as well as nucleotides which are units of DNA and RNA, or the units. In the present specification, the “oligonucleotide derivative” means a polymer chain in which such nucleotide derivatives are linked, but the length (number of units) is not particularly limited. In addition, although not specifically exemplified, for example, spermine and poly (L-lysine) -graft-dextran (Bioconjugate Chem. 1997, 8, 3., Bioconjugate Chem. 1998, 9, 292.) etc. In addition, the target nucleic acid may be detected.

  Hereinafter, embodiments of the present invention will be described in detail.

(Fluorescently labeled oligonucleotide derivative)
The fluorescently labeled oligonucleotide derivative of the present invention includes a single-stranded polymer chain including at least a nucleotide or a derivative thereof as a first monomer unit, and the polymer chain includes three second monomer units including a fluorescent dye. In addition to the above, at least one of the first monomer units can be provided between these second monomer units.

(First monomer unit)
The polymer chain comprises a first monomer unit. The first monomer unit has a structure based on nucleotides or derivatives thereof. That is, the first monomer unit can have, for example, a polymer chain composed of a basic skeleton such as DNA, modified DNA, RNA, modified RNA, LNA, SNA, aTNA, PNA, or other basic skeleton unit structure. Such a basic skeleton may be, for example, a unit structure composed of a phosphate-alkylene chain that is preferably used in the second monomer unit described later. The first monomer unit is typically a deoxynucleotide, a ribonucleotide or the like. The unit structure of the first monomer unit may be one type or two or more types, but is preferably a single unit structure.

  The first monomer unit is a base complementary to the base in the target sequence, typically the natural bases A (adenine), T (thymine), G (guanine), U (uracil) and C (cytosine). Alternatively, it can have analogs thereof. When the first monomer unit has such a base, a complementary base sequence that hybridizes with the target sequence can be formed in the polymer chain formed by polymerization of the first monomer unit. Examples of the base include A (adenine), T (thymine), G (guanine), U (uracil), and C (cytosine), as well as known base analogs having the same base pairing ability as each of these. May be.

  A base sequence for hybridizing with the target sequence is formed by the sequence of the base and the like in the first monomer unit. The length of the base sequence for hybridization (number of first monomer units) is not particularly limited, but is preferably 10 mer or more and 60 mer or less. More preferably, it is 15 mer or more. Further, it is more preferably 30 mer or less, further preferably 25 mer or less, and further preferably 20 mer or less.

  The polymer chain is preferably composed mainly of such a first monomer unit, and the length thereof is not particularly limited, but is preferably 10 mer or more and 60 mer or less. More preferably, it is 15 mer or more. More preferably, it is 30 mer or less.

  The polymer chain is a single chain at least when subjected to hybridization. This is because, when it is single-stranded, the fluorescent dye in the second monomer unit can self-associate and be quenched at the time of non-hybridization. If necessary, before the hybridization, the complementary strand may be prepared, distributed, stored, etc. as a double strand.

(Second monomer unit)
The polymer chain includes three or more second monomer units including a fluorescent dye, and at least one of the first monomer units may be included between the second monomer units. By configuring the arrangement of the second monomer unit and the first monomer unit in this way, when the polymer chain is not hybridized, the fluorescent dyes in the second monomer unit are associated with each other to be self-quenched. In hybridization, fluorescence can be effectively generated in the target nucleic acid and the hybridized product.

  It is preferable that all the second monomer units in the polymer chain are provided in a portion (hybridization sequence) designed to hybridize with the target sequence in the polymer chain. By doing so, the fluorescence intensity by the fluorescent dye in the second monomer unit can be effectively increased.

  When three or more second monomer units are provided, quenching can be effectively performed and sufficient fluorescence can be generated. When the number of the second monomer units is 2 or less, quenching is not sufficiently performed. It is preferable that four or more second monomer units are provided. When the number of fluorescent dyes increases, the fluorescence intensity tends to be increased. When the number of fluorescent dyes is four or more, the hybridization is performed in a state where the fluorescence intensity is mismatched when hybridized in a completely complementary state with the target sequence. This is because it is larger than the fluorescence intensity at the time. Further, the upper limit of the number of second monomer units is not particularly limited, but is appropriately determined according to the length of the hybridization sequence.

  At least one first monomer unit is provided between the second monomer units. By arranging the first monomer unit between the second monomer units, both self-quenching and light emission can be achieved. More preferably, it is two or more. By interposing two first monomer units, self-quenching is effectively performed and there is fluorescence in which the emission intensity is enhanced. On the other hand, the number of intervening first monomer units is preferably 4 or less. This is because the self-quenching efficiency between the dyes decreases when the number is 5 or more.

  The second monomer unit is additionally introduced into the polymer chain portion constituting the hybridization sequence. That is, instead of replacing a specific first monomer unit in the hybridization sequence, it is added to a predetermined number of first monomer units constituting the hybridization sequence. Since the second monomer unit can be additionally introduced to the hybridizing sequence composed of the first monomer unit, the polymer chain can be inserted with a high degree of freedom regardless of the length of the hybridizing sequence and the constituent bases. Two monomer units can be provided, and the intervening state of the first monomer unit can be freely adjusted. For this reason, the degree of quenching and the intensity of fluorescence can be controlled.

  The second monomer unit can have a basic skeleton that can be applied to the first monomer unit. That is, the structure excluding the base of the basic skeleton unit that forms DNA, modified DNA, RNA, modified RNA, LNA, PNA, SNA, aTNA, and other known hybridized products can be included as a unit structure. Unlike the first monomer unit, the second monomer unit has a fluorescent dye instead of a base or the like. The basic skeleton of the second monomer unit may be different from or the same as the first monomer unit. The second monomer unit preferably has a unit structure mainly composed of an alkylene chain (R1) represented by the following formula (1). In the formula (1), A1 represents a nucleotide derivative or an oligonucleotide derivative that is linked via a hydrogen atom on the 5 ′ side of the oligonucleotide derivative, a phosphate group, or a phosphodiester bond, and B1 represents 3 of the oligonucleotide derivative. 'Represents a nucleotide derivative or an oligonucleotide derivative linked via a hydrogen atom on the side, a phosphate group, or a phosphodiester bond. Preferably, it represents a nucleotide derivative or an oligonucleotide derivative in which at least one of A1 and B1 is linked via a phosphate group or a phosphodiester bond.

  In the formula (1), X represents a fluorescent dye. The fluorescent dye is not particularly limited, and a known fluorescent dye can be appropriately selected and used as described later. Preferably, it is a fluorescent dye having a condensed aromatic ring system. Typically, X has a perylene group which may be substituted. The perylene group is advantageous in that it is photochemically stable and has high quantum efficiency. In the perylene group, hydrogen atoms bonded to one or more carbon atoms other than carbon atoms connected to Z or an appropriate linking group may be appropriately substituted. Although the substituent is not particularly limited, for example, each independently a hydrogen atom; an alkyl group or alkoxy group having 1 to 20 carbon atoms that is unsubstituted or substituted with a halogen atom, a hydroxyl group, an amino group, a nitro group, or a carboxyl group An unsubstituted or substituted alkenyl or alkynyl group having 2 to 20 carbon atoms substituted with a halogen atom, hydroxyl group, amino group, nitro group or carboxyl group; hydroxyl group; halogen atom; amino group; nitro group; or carboxyl group be able to.

  In the formula (1), R1 represents an alkylene chain which has 2 or 3 carbon atoms and may be substituted. R2 represents an alkylene chain which has 0 to 2 carbon atoms and may be substituted. R2 is preferably bonded to the second carbon atom from the oxygen atom on the 5 'side of the alkylene chain of R1.

  In the formula (1), Z represents a direct bond or a linking group. Examples of Z as a linking group include —NHCO—, NHCS—, CONH—, —O—, and the like, or those containing these groups. In addition, in the connection part (dotted line part) to a connection group, 1 selected from the phenylene group, the alkylene group, the alkenylene group, and the alkynylene group which may be substituted in consideration of the relationship with the size of the fluorescent dye. You may interpose a seed | species or 2 or more types in combination. In addition, the compound used as a fluorescent label may be appropriately derivatized in order to be linked to the unit represented by the formula (1). Moreover, the atom on the fluorescent label connected with respect to the unit represented by Formula (1) is not specifically limited.

  As the substituent of R1 to R2 in the formula (1), it is unsubstituted or substituted with a halogen atom, a hydroxyl group, an amino group, a nitro group, a carboxyl group, or the like, preferably 1 to 10, and more preferably 1 to 10. 1 to 4 alkyl groups or alkoxy groups; 2-20 carbon atoms, preferably 2-10, more preferably 2-4, which are unsubstituted or substituted with halogen atoms, hydroxyl groups, amino groups, nitro groups, carboxy groups or the like. An alkenyl group or alkynyl group; a hydroxyl group, a halogen atom, an amino group, a nitro group, or a carboxy group. 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 from the 5 'oxygen to the second or third carbon atom.

  Regarding the plurality of second monomer units, the number of carbon atoms of the alkylene chain in R 1 in the formula (1) may be the same or different.

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

  Examples of the second monomer unit represented by the formula (1) include units represented by the following formulas.

  Furthermore, as a unit represented by Formula (1), what is represented by the following formula | equation (2) and (3) is mentioned. In these formulas, the same symbols as in formula (1) are synonymous with those in formula (1). In addition, although X represents a fluorescent dye similarly to Formula (1), -L2-L1-X and -L4-L3-X in each formula can also be regarded as a fluorescent chromophore, respectively.

  In formula (2), L1 is an alkynylene group having 2 to 4 carbon atoms. Alkynylene groups include ethynylene groups (—C≡C—), propynylene groups (—C≡C—C—, —C—C≡C—), butynylene groups (—C≡C—C—C—, —C). -C≡C-C-, -C-C-C≡C-). Preferably, it is an ethynylene group.

  In the formula (2), L2 represents a phenylene group. The phenylene group may not be substituted, but a hydrogen atom bonded to one or more carbon atoms not involved in the connection may be substituted. Although the substituent is not particularly limited, for example, each independently a hydrogen atom; an alkyl group or alkoxy group having 1 to 20 carbon atoms that is unsubstituted or substituted with a halogen atom, a hydroxyl group, an amino group, a nitro group, or a carboxyl group An unsubstituted or substituted alkenyl or alkynyl group having 2 to 20 carbon atoms substituted with a halogen atom, hydroxyl group, amino group, nitro group or carboxyl group; hydroxyl group; halogen atom; amino group; nitro group; or carboxyl group be able to.

  Although the connection bonding site in the phenylene group of L2 is not particularly limited, it may be connected to the main chain at any of the ortho, meta and para positions from the connection position with respect to R1. The para position is preferred.

  X in the formula (2) represents a fluorescent dye as in the formula (1), and is not particularly limited as in the formula (1), and a known fluorescent dye can be appropriately selected and used as described later. , Preferably a fluorescent dye having a condensed aromatic ring system, and typically X has an optionally substituted perylene group.

  The connecting position of L1 to the fluorescent dye X is not particularly limited. For example, when the fluorescent dye is perylene, it may be any of the 1-position, 2-position, 3-position, etc. of perylene. Preferably, it is any one of the 2nd, 4th, 9th and 10th positions.

  In the formula (3), X represents a fluorescent dye, and is not particularly limited as in the formula (1). A known fluorescent dye can be appropriately selected and used as described later, and preferably a condensed fragrance is used. A fluorescent dye of the group ring system, typically, X has a perylene group which may be substituted.

  In Formula (2), L3 is an alkynylene group having 2 to 4 carbon atoms. Alkynylene groups include ethynylene groups (—C≡C—), propynylene groups (—C≡C—C—, —C—C≡C—), butynylene groups (—C≡C—C—C—, —C). -C≡C-C-, -C-C-C≡C-). Preferably, it is an ethynylene group.

  The connecting position of L3 to the fluorescent dye X is not particularly limited. For example, when the fluorescent dye is perylene, it may be any of the 1-position, 2-position, 3-position, etc. of the perylene group. Preferably, it is any one of the 2nd, 4th, 9th and 10th positions.

  In Formula (2), L4 is an alkylene group having 1 to 3 carbon atoms. For example, methylene group, ethylene group, and propylene group. Preferably, it is an ethylene group.

  In Formula (2) and Formula (3), A1 represents a nucleotide derivative or oligonucleotide derivative linked via a hydrogen atom on the 5 ′ side of the oligonucleotide derivative, a phosphate group, or a phosphodiester bond, and B1 is This represents a nucleotide derivative or oligonucleotide derivative that is linked via a hydrogen atom on the 3 ′ side of an oligonucleotide derivative, a phosphate group, or a phosphodiester bond. Nucleotides and oligonucleotides have a phosphate group at the carbon atom at the 5 'position of the 5 monosaccharide. When B1 is a nucleotide and an oligonucleotide, they are linked by a phosphodiester bond through their phosphate group.

  Examples of the second monomer unit represented by the formulas (2) and (3) include units represented by the following formulas.

(Fluorescent dye)
The fluorescent dye is not particularly limited as long as it is self-associating and quenching when the fluorescent oligonucleotide is single-stranded, and emits fluorescence when the fluorescent oligonucleotide is hybridized with a complementary strand such as DNA. Examples of fluorescent dyes include cyanine dyes, merocyanine dyes, acridine dyes, coumarin dyes, ethidium dyes, flavin dyes, condensed aromatic ring dyes, xanthene dyes, and the like. Can be used. More specifically, cyanine dyes, coumarin dyes, ethidium dyes, condensed aromatic ring dyes, and xanthene dyes can be preferably used. More specifically, it is selected from the group consisting of pyrene, perylene and derivatives thereof which are condensed aromatic ring dyes. The condensed aromatic ring dye is hydrophobic, easily self-associates, has a large quenching effect, and tends to emit strong fluorescence by intercalating into a double strand with a complementary strand.

  The fluorescent dyes in the second monomer unit may be the same as or different from each other. For example, in the second monomer unit having different fluorescent dyes, these fluorescent dyes may be used as two fluorescent dyes (donor (donor) and acceptor (acceptor)) that generate energy transfer by resonance. it can. By adjoining these fluorescent dyes with one or more first monomer units interposed, FRET can be generated between these two fluorescent dyes upon hybridization with the target nucleic acid. Examples of the combination of the second monomer units provided with these two fluorescent dyes include the following combinations.

As described above, the fluorescently labeled oligonucleotide derivative of the present invention includes three or more second monomer units, with one or more first monomer units arranged between the second monomer units. It is configured. The appropriate number of second monomer units, the fluorescent labeling dye in the second monomer unit, the number of first monomer units between the second monomer units, and the like can be determined as appropriate. That is, the fluorescently labeled oligonucleotide derivative of the present invention is
(A) When non-hybridized with the target nucleic acid, they associate with each other and are self-quenched.
(B) Upon hybridization with the target nucleic acid, fluorescence is emitted by the cancellation of the association.
So that the second monomer units are separated from each other by the first monomer units so as to be provided in the polymer chain.

(Method for producing fluorescently labeled oligonucleotide derivative)
Such fluorescently labeled oligonucleotide derivatives can be produced by known methods. For example, when the polymer chain of the fluorescently labeled oligonucleotide derivative is DNA, RNA, PNA, PNA or the like, it is well known to those skilled in the art to synthesize such a polymer chain using an amidite derivative corresponding to the first monomer unit. . That is, when the polymer chain is a polymer chain by a phosphate ester bond such as a nucleotide, the polymer chain is an amidite derivative corresponding to a nucleotide or a derivative thereof as a first monomer unit in a normal nucleic acid solid phase synthesis method. And an amidide derivative capable of introducing the second monomer unit into the polymer chain.

  Amidite derivatives for the second monomer unit having a phosphoric acid-alkylene chain represented by the formula (1) as unit structures are aminoalkyl diols such as D-threonyl and 3-amino1,2-propanediol. Is protected with an appropriate protecting group such as an allyloxycarbonyl group, one hydroxyl group is protected with dimethoxytrityl chloride and the like, and then the other hydroxyl group is 2-cyanoethyl N, N, N, N-tetraisopropyl. Phosphorodiamidide is introduced to obtain an amidite derivative. Then, a fluorescent dye may be introduced into this amidite derivative to form an amidite monomer. Condensed aromatic ring-based perylene compounds include, for example, the method described in Nature Protocols, Vol. 2, pages 203-212, Journal of the American Chemical Society (Nature Protocols, 2007). The method described in Journal of the American Chemical Society, 2003, 125, 2217-2223, and the method described in Tetrahedron Letters, 2007, 48, 6759-6762 can be applied.

  The fluorescent dye may be introduced after synthesis of an oligonucleotide having the above amidite derivative unit 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 having a carboxylic acid group or an isocyanate group introduced or retained so as to be reactive with the amino group A fluorescent dye may be introduced by reaction.

  Moreover, as a method of acquiring the amidite derivative corresponding to the 2nd monomer unit represented by Formula (2) and Formula (3), the following schemes are respectively illustrated, for example.

  In the above scheme, the amino group of D-threoninol is protected with ethyl trifluoroacetate, and then DMT is introduced and deprotected to obtain a DMT derivative of D-threoninol. Thereafter, this DMT derivative and 4-pentynoic acid are introduced into the amino group of D-threoninol to obtain a pentynoic acid-DMT derivative. A separately synthesized halogen (here Br) perylene is reacted with this DMT derivative to introduce perylene via an ethynylene group to obtain an ethynylperylene DMT derivative. This ethynylperylene DMT derivative is then amidite-ized.

  In the above scheme, after synthesizing 4-ethynylbenzoic acid, a DMT derivative of D-threoninol is introduced into the hydroxyl group of the carboxyl group to obtain a phenylethynyl DMT derivative. Thereafter, it is reacted with a halogenated perylene to introduce perylene via an ethynylene group to obtain a phenylethynylperylene DMT derivative. Next, this phenylethynylperylene DMT derivative is amidated.

  Although not illustrated, after obtaining an amidite derivative derived from D-threoninol, the first monomer unit or the second monomer unit may be formed using the amidite derivative to form a final amidite derivative.

  After obtaining the amidite derivatives and the like corresponding to the first monomer unit and the second monomer unit, a conventionally known DNA synthesis method, for example, Nature Protocols, 2007, Vol. 2, pages 203 to 212, is used. According to the method described in 1), an oligonucleotide having the first unit or the second unit at a desired site can be synthesized.

(Use of fluorescently labeled oligonucleotide derivatives)
The fluorescently labeled oligonucleotide derivative of the present invention can be used for various applications. Typically, it is a probe, but more specifically, (1) a probe used in an assay in a liquid phase and a kit including such a probe, (2) DNA using PCR such as real-time PCR and TaqMan probe (3) as a probe for amplification and a kit comprising such a probe, (3) a capture probe such as a DNA array, an immobilized body comprising the fluorescently labeled oligonucleotide derivative of the present invention as the capture probe, and a kit comprising such an immobilized body (4) ) An immobilized body in which the fluorescently labeled oligonucleotide derivative of the present invention is immobilized on a solid phase body such as a bead, fiber or hydrogel, and a kit comprising such an immobilized body; (5) detection of target nucleic acid in cells or tissues; Probes for tracking and kits with such probes, and (6) fluorescence in situ It can be used as a kit or the like comprising a Lee hybridization (FISH) probe and such probes.

(Target nucleic acid detection method)
The method for detecting a target nucleic acid of the present invention comprises a hybridization step in which the fluorescently labeled oligonucleotide derivative of the present invention and a nucleic acid sample possibly containing the target nucleic acid are brought into contact with each other so as to be capable of hybridizing, and the fluorescent dye is detected. And a detection step for detecting a hybridization product of the fluorescently labeled oligonucleotide derivative and the target nucleic acid in the hybridization step under possible conditions. According to this detection method, hybridization with a target nucleic acid can be detected effectively by the self-quenching property at the time of non-hybridization and the light emission property at the time of hybridization provided in the fluorescently labeled oligonucleotide derivative of the present invention. . Further, unlike conventional molecular beacons and the like, the target nucleic acid can be detected with high responsiveness because the stem strand is not released and the fluorescent dye does not inhibit hybridization with the target sequence.

  The embodiment of the hybridization step is not particularly limited. The hybridization process includes (1) detection, identification, quantification, etc. of target nucleic acid in liquid phase, 2) detection, identification, quantification, etc. of target nucleic acid by DNA amplification using real-time PCR, PCR such as TaqMan probe, etc. 3) Detection, identification, quantification, etc. of target nucleic acids using DNA arrays, etc. (4) Detection, identification, quantification, etc. of target nucleic acids using immobilized bodies using beads, etc., (5) In cells and tissues It can be used in various forms such as detection, identification and quantification of target nucleic acid, and (6) detection, identification and quantification of target nucleic acid by fluorescence in situ hybridization (FISH). Therefore, also in the hybridization step, the fluorescently labeled oligonucleotide derivative of the present invention can be used in a form such as the above-described use.

  Moreover, the embodiment of the detection step is not particularly limited. The detection of the hybridized product in the detection step using a fluorescent dye may be performed at a fluorescent wavelength that is planned in advance based on the structure of the fluorescently labeled oligonucleotide derivative used. In addition to detection and identification of the target nucleic acid, the target nucleic acid can be quantified based on the fluorescence intensity.

  Hereinafter, specific examples embodying the present invention will be described. However, the present invention is not limited to the following specific examples.

(Synthesis of an oligonucleotide derivative having a monomer unit E having a perylene fluorescent dye)
1-1. Synthesis of amidite monomer corresponding to synthesis of DNA containing fluorescent dye E DNA containing fluorescent dye E was synthesized according to the following scheme 1-1.

Compound 1-1 was synthesized by the method described in Chemical Communications, 2011, 47, 6404-6406. That is, compound 1-1 (0.70 g, 0.951 mmol) and Pd (OH) ₂ (180 mg) were placed in a two-necked flask, dissolved in about 60 ml of dry THF, and purged with nitrogen. Next, the flask was once evacuated, purged with hydrogen, and stirred. After completion of the reaction, Pd (OH) ₂ was filtered through Celite, evaporated, and dried in vacuo. This was purified by silica gel column chromatography (developing solvent composition: chloroform: hexane: ethyl acetate: triethylamine = 30: 30: 30: 2.7) to obtain compound 1-2 (0.48 g: 0.65 mmol, yield 68%). Obtained.

  Compound 1-2 (0.48 g: 0.65 mmol, 1.0 eq) was placed in an eggplant flask, purged with nitrogen, dissolved in dehydrated methylene chloride and dehydrated acetonitrile, azeotroped twice, and then about 3.0 ml of dehydrated methylene chloride and dehydrated acetonitrile. It was dissolved in about 3.0 ml and triethylamine about 0.45 ml. Subsequently, 2-cyanoethyldiisopropylchlorophosphoramidite (0.31 g, 0.293 mmol, 2.1 equivalents) was slowly added dropwise on an ice bath and stirred. After 20 minutes, the ice bath was removed and the mixture was allowed to react at room temperature for about 40 minutes to obtain Compound 1-3. The DNA was introduced in accordance with the method described in Chemical Communications, 2011, 47, 6404-6406.

(Measurement of fluorescence intensity change due to duplex formation with target sequence)
A target nucleic acid including an oligonucleotide finally synthesized using an amidite derivative having each base and a target sequence (b3a2) and a partial target nucleic acid partially matching the target sequence (b2a2)) are shown below.
(Fluorescently labeled oligonucleotide)
1E 5 'AAGGGCTETTTGAACTC 3' (SEQ ID NO: 1)
2E-2 5 'AAGGGCTETTETGAACTC 3' (SEQ ID NO: 2)
3E-2 5 'AAGGGECTETTETGAACTC 3' (SEQ ID NO: 3)
4E-2 5 'AAGGGECTETTETGEAACTC 3' (SEQ ID NO: 4)
5E-2 5 'AAGEGGECTETTETGEAACTC 3' (SEQ ID NO: 5)
6E-2 5 'AAGEGGECTETTETGEAAECTC 3' (SEQ ID NO: 6)

(Target nucleic acid and partial target nucleic acid)
b3a2: 3'-AC TTCCCGAAAACTTGAG AC-5 '(including target sequence completely complementary to oligonucleotide) (SEQ ID NO: 7)
b2a2: 3'-AC TTCCCGAA GAAGGAATA-5 '(including a sequence partially complementary to the oligonucleotide) (SEQ ID NO: 8) (probe is complementary to the underlined portion)

  Using these oligonucleotides, the fluorescence intensity of each fluorescence intensity in the temporary heavy chain state of the fluorescently labeled oligonucleotide and the double strand state with the target nucleic acid, etc. is measured, and the S / B ratio (in the double strand state with b3a2) (Fluorescence intensity / fluorescence intensity in oligonucleotide single-stranded state)

The measurement conditions in the single chain state were as follows.
Oligonucleotide concentration 1 μM, pH 7.0 (10 mM phosphate buffer), [NaCl] = 100 mM, 20 ° C, 425 nm excitation, 460 nm fluorescence intensity measured

The measurement conditions in the double chain state were as follows.
Oligonucleotide concentration 1 μM, b3a2 concentration 2 μM, pH 7.0 (10 mM phosphate buffer), [NaCl] = 100 mM, 20 ° C, 425 nm excitation, 460 nm fluorescence intensity measured

  The results are shown in the table below.

  As described above, the fluorescence intensity in the single-stranded state decreased as the number of fluorescent dyes E introduced was increased, and drastically decreased particularly with three or more. On the other hand, when the target (b3a2) was double-stranded, the fluorescence intensity increased as the number of fluorescent dyes E increased. As a result, the signal / background ratio (S / B ratio) improved as E increased, and a high S / B ratio could be achieved with a probe in which three or more fluorescent dyes E were introduced.

  In addition, FIG. 2 and FIG. 3 show the single-stranded fluorescence spectrum of 4E-2, the fluorescence spectrum when b3a2 is combined with a double strand, and the fluorescence spectrum in the double-stranded state with b2a2.

  As shown in FIG. 2 and FIG. 3, the fluorescence intensity at 460 nm is 467.5 and 12.6, respectively, when b3a2 is double-stranded and when partially complementary b2a2 is double-stranded. There was a difference of nearly 40 times. Thus, according to the fluorescently labeled oligonucleotide derivative of the present invention, it was found that the probe of the present invention has sufficient sequence specificity.

(Synthesis of fluorescently labeled oligonucleotide derivative comprising monomer unit E ′ comprising perylene fluorescent dye)
In this example, a fluorescently labeled oligonucleotide derivative having monomer units below the fluorescent dye was synthesized.

  Oligonucleotide derivatives having the above-mentioned three monomer units and having the following sequences were synthesized by the method described in Tetrahedron Letters, 2007, Vol. 48, pages 6759-6762. A target nucleic acid Comp1 was prepared with the following sequence.

(Fluorescently labeled oligonucleotide)
3E'-2: 5'-GGTE'TAE'TTE'ATGCCG-3 '(SEQ ID NO: 9)
(Target nucleic acid)
Comp1: 3′-CCAATAATACGGCCTG-5 ′ (SEQ ID NO: 10)

  The result of excitation of this oligonucleotide (3E'-2) at 425 nm under the conditions of oligonucleotide concentration 1 μM, pH 7.0 (10 mM phosphate buffer), NaCl concentration 100 mM, 20 ° C. and the corresponding target nucleic acid Comp1 FIG. 4 shows the result of measuring the fluorescence intensity at 460 nm under the same conditions in addition to the concentration of 2 μM.

  As shown in FIG. 4, the fluorescence intensity at 460 nm in the single-stranded state was 18.9, whereas the fluorescence intensity in the double-stranded state was 467. Thus, by introducing three perylene fluorescent dyes E ', a high sensitivity with an S / B ratio of 24.7 could be realized.

(Synthesis of fluorescently labeled oligonucleotide derivative using monomer unit F provided with perylene fluorescent dye)
In this example, fluorescently labeled oligonucleotide derivatives having the following monomer units were synthesized.

An oligonucleotide derivative having four monomer units and having the following sequence was synthesized according to the method described in Chemical Communications, 2011, 47, 6404-6406. A target nucleic acid Comp2 was prepared with the following sequence.
(Fluorescently labeled oligonucleotide)
4F0L: 5'-AGG-F-TA-F-TCGC-F-AA-F-TCC-3 '(SEQ ID NO: 11)
(Target nucleic acid)
comp2: 3′-TCCATAGCGTTAGG-5 ′ (SEQ ID NO: 12)

  FIG. 5 shows fluorescence spectra in a single-stranded state of this oligonucleotide and in a double-stranded state with the target nucleic acid. As shown in FIG. 5, when the oligonucleotide was excited at 440 nm under the conditions of oligonucleotide concentration 1 μM, pH 7.0 (10 mM phosphate buffer), NaCl concentration 100 mM, 20 ° C., the fluorescence intensity at 478 nm was 11.8. On the other hand, when the corresponding target nucleic acid comp2 was added to a concentration of 1.2 μM and the fluorescence intensity at 478 nm was measured under the same conditions, it was 528. By using the monomer unit F of the present invention, a high sensitivity with an S / B ratio of 44.7 could be realized.

(Synthesis of oligonucleotide derivatives having both monomer units F and L introduced)
In this example, a fluorescently labeled oligonucleotide derivative having monomer unit F and monomer unit L shown below was synthesized. In addition, target nucleic acid Comp2 was prepared.

  An oligonucleotide derivative having four monomer units and having the following sequence was synthesized according to the method described in Chemical Communications, 2011, 47, 6404-6406. In this case, since the emission spectrum of the fluorescent dye f in the monomer unit F and the absorption spectrum of the fluorescent dye l in the monomer unit L overlap, only the fluorescent dye l is obtained by fluorescence resonance energy transfer (FRET) even when the fluorescent dye f is excited. Fluorescence was observed.

(Fluorescently labeled oligonucleotide)
4F1L: 5'-AGG-F-TA-F-TCLGC-F-AA-F-TCC-3 '(SEQ ID NO: 13)

  FIG. 6 shows fluorescence spectra in a single-stranded state of this oligonucleotide and in a double-stranded state with the target nucleic acid. As shown in FIG. 6, when excited at 440 nm under the conditions of oligonucleotide concentration 1 μM, pH 7.0 (10 mM phosphate buffer), NaCl concentration 100 mM, 20 ° C., the fluorescence intensity at 503 nm was 7.81. . On the other hand, the corresponding target nucleic acid Comp2 (3'-TCCATAGCGTTAGG-5 ') was added to a concentration of 1.2 μM, and the fluorescence intensity at 503 nm was measured under the same conditions. As a result, it was 183. Thus, even when different fluorescent dyes were introduced, a high sensitivity with an S / B ratio of 23.4 could be realized. It was also found that FRET can be realized.

SEQ ID NOs: 1 to 13: fluorescently labeled oligonucleotides

Claims (7)

A fluorescently labeled oligonucleotide derivative comprising:
Comprising a single-stranded polymer chain comprising at least a nucleotide or a derivative thereof as a first monomer unit;
Including three or more second monomer units with a fluorescent dye in the polymer chain;
Between these second monomer units, 2 or more and 4 or less of the first monomer units are provided,
, Oligonucleotide derivatives.   The oligonucleotide derivative according to claim 1, wherein at least 4 or more of the second monomer units are provided in the polymer chain. The oligonucleotide derivative according to claim 1 or 2 , wherein the second monomer unit is represented by the following formula (1).

(In the formula, X represents a fluorescent dye, R 1 represents an alkylene chain having 2 or 3 carbon atoms and may be substituted. R 2 has 0 to 2 carbon atoms. Represents an alkylene chain which may be substituted, Z represents a direct bond or a linking group, and A1 is linked via a hydrogen atom on the 5 ′ side of an oligonucleotide derivative, a phosphate group, or a phosphodiester bond. Represents a nucleotide derivative or an oligonucleotide derivative, and B1 represents a nucleotide derivative or an oligonucleotide derivative linked via a hydrogen atom on the 3 ′ side of the oligonucleotide derivative, a phosphate group, or a phosphodiester bond. The oligonucleotide derivative according to any one of claims 1 to 3 , further comprising two or more second monomer units each having two fluorescent dyes that cause energy transfer by resonance. The oligonucleotide derivative according to any one of claims 1 to 4 , wherein the fluorescent dye is a condensed ring dye. An immobilized body in which the fluorescently labeled oligonucleotide derivative according to any one of claims 1 to 5 is immobilized on a solid phase body. A method for detecting a target nucleic acid, comprising:
A hybridization step of bringing the fluorescently labeled oligonucleotide derivative according to any one of claims 1 to 5 and a nucleic acid sample possibly containing a target nucleic acid into contact with each other so as to be hybridizable;
A detection step for detecting a hybridization product of the fluorescently labeled oligonucleotide derivative and the target nucleic acid in the hybridization step under conditions where the fluorescent dye can be detected;
A detection method comprising:

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