JP2011135823A - Oligonucleotide probe and use of the same - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a fluorescent oligonucleotide probe which has high design freedom and high versatility. <P>SOLUTION: The oligonucleotide probe forms a stem and a loop, and comprises at least one fluorophore as shown in formula (1) which is arranged between adjacent nucleotides of the stem and at least one quencher connected to a unit arranged in a region corresponding to the at least one fluorophore between the adjacent nucleotides of the stem. In the formula, X represents the fluorophore, R1 represents an alkylene chain whose carbon number is 2 or 3 and which may be substituted, R2 represents an alkylene chain whose carbon number is 0 to 2 and which may be substituted, and Z represents direct coupling or a linking group. <P>COPYRIGHT: (C)2011,JPO&INPIT

Description

  The present invention relates to an oligonucleotide probe and use thereof.

  Oligonucleotide probes include probes that self-generate fluorescence. Molecular beacons are known as such fluorescent self-generated probes. A molecular beacon is an oligonucleotide that emits a detectable fluorescent signal when hybridized with a target nucleic acid (Patent Document 1). Molecular beacons are artificial oligonucleotides having a stem-loop structure and having a fluorescent substance (fluorophore) and a fluorescent coloring inhibitor (quencher) at both ends. In the molecular beacon, normally, in a single-stranded state, the stem region forms a double strand, and as a result of the fluorophore and quencher introduced at both ends being close to each other, the fluorescence of the fluorophore is quenched by the quencher. On the other hand, when hybridizing with an oligonucleotide complementary to the base sequence of the loop region in the probe, the fluorophore and the quencher are separated and a fluorescent signal is emitted. By using such a molecular beacon as a probe, hybridization with a target nucleic acid can be detected without fluorescently labeling the target.

  Conventionally, this type of molecular beacon is mainly used for a liquid phase reaction, for example, for detecting an amplification product in real-time PCR or the like.

JP 2004-329209 A

  The molecular beacon forms a stable stem at a temperature sufficiently lower than the melting temperature of the stem and quenches. However, when the target nucleic acid is present, the stem is opened by hybridization with the target nucleic acid and emits fluorescence based on the fluorophore. However, the behavior and color intensity with such a target nucleic acid greatly vary depending on the temperature. That is, when a target nucleic acid is present under a temperature lower than the melting temperature of the stem, the molecular beacon forms a double strand with the target nucleic acid and emits fluorescence, but when the temperature rises, the molecular beacon moves away from the target nucleic acid. It will be extinguished. Therefore, in order to secure a quenching state in the absence of the target nucleic acid and reduce the background, it is necessary to raise the stem melting temperature sufficiently to form a stable quenching state. At the same time, in order to more reliably detect hybridization between the target nucleic acid and the molecular beacon, it is preferable to increase the melting temperature of the molecular beacon and the target nucleic acid as close as possible to the melting temperature of the stem.

  However, since the length of the entire molecular beacon is limited to some extent, if the stem is lengthened to increase the melting temperature of the stem, the probe design freedom may be reduced and the detection specificity of the target nucleic acid may be reduced. .

  In particular, when considering a molecular beacon array in which molecular beacons to be detected corresponding to various types of target nucleic acids are immobilized on a solid phase carrier, in order to ensure detection accuracy, the melting temperature of the stem is increased. It is preferable to design such that it can hybridize with a sufficiently long target nucleic acid. However, for the same reason as described above, it has been difficult to simultaneously realize both of these with a molecular beacon.

  That is, with the conventional molecular beacon, it is extremely difficult to ensure the specificity for the target nucleic acid while suppressing the background.

  Therefore, an object of the present invention is to provide a fluorescent oligonucleotide probe with higher design flexibility and versatility and use thereof.

  The present inventors do not quench or emit light largely depending only on the proximity effect caused by the formation of the stem and the separation effect caused by the formation of the double strand with the target nucleic acid, but are stable when approached by the formation of the stem. It was found that by arranging the fluorophore and the quencher so that a stack structure can be formed, quenching can be performed more reliably and stem thermal stability can be improved. In addition, it has been found that there is a case where luminescence can be enhanced by placing the fluorophore in the duplex with the target nucleic acid when the duplex with the target nucleic acid is formed. Based on these findings, the present inventors have completed the present invention. According to the disclosure of the present specification, the following means are provided.

According to the disclosure of the present specification, an oligonucleotide probe capable of forming a stem and a loop, which is connected to a unit represented by the following formula (1) arranged between adjacent nucleotides of the stem: One fluorophore,
(In the formula, X represents a fluorophore, 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. And Z represents a direct bond or a linking group.
At least two quenchers connected to a unit represented by the following formula (2) arranged at a site capable of pairing with the at least one fluorophore between adjacent nucleotides of the stem;
(In the formula, Y represents a quencher, 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.)
A probe is provided. In the above formulas (1) and (2), the phosphate group (PO ₃ ⁻ ) in the phosphodiester bond may be non-dissociated (PO ₃ H).

  In the oligonucleotide probe disclosed in the present specification, the fluorophore is preferably arranged inside a nucleotide chain constituting a base sequence that specifically hybridizes with a target nucleic acid. In the oligonucleotide probe disclosed in the present specification, the fluorophore is any one selected from the group consisting of a cyanine dye, a merocyanine dye, a condensed aromatic ring dye, and a xanthene dye, and the quencher is And any one selected from the group consisting of azo dyes. More preferably, the fluorophore is any one selected from the group consisting of Cy3, Cy5, thiazole orange, oxazole yellow, perylene, fluorescein, rhodamine, tetramethylrhodamine and Texas red, and the quencher is methyl It is preferably any one selected from red, azobenzene and methylthioazobenzene.

  The fluorophore is a condensed aromatic ring dye, the quencher is preferably anthraquinone or a derivative thereof, the fluorophore is perylene, and the quencher is more preferably anthraquinone.

  Moreover, it is preferable that the oligonucleotide probe of this specification can form one said stem and one said loop.

  According to the disclosure of the present specification, a probe-immobilized body for detecting a target nucleic acid, which has a base sequence capable of specifically hybridizing with a solid phase carrier and at least a part of the target nucleic acid, An immobilized body comprising any of the above oligonucleotide probes held on the solid phase carrier is provided. In the probe-immobilized body disclosed in the present specification, the solid phase carrier is preferably a plate-like body. Moreover, it is preferable that the probe fixed body disclosed in this specification includes a plurality of oligonucleotide probes capable of detecting a plurality of target nucleic acids in an array. In addition, the probe-immobilized body disclosed in the present specification is preferably used for detection of SNPs or gene mutations.

  According to the disclosure of the present specification, there is provided a method for detecting a target nucleic acid, the oligonucleotide probe disclosed in the present specification, having a base sequence capable of specifically hybridizing with at least a part of the target nucleic acid. A method is provided that uses an oligonucleotide probe to detect a signal based on the fluorophore of the oligonucleotide probe.

  In the detection method disclosed in the present specification, it is preferable to use an immobilized body in which a plurality of oligonucleotide probes capable of hybridization corresponding to a plurality of target nucleic acids are held on a solid phase carrier.

It is a figure explaining the outline | summary of the oligonucleotide probe of an indication of this specification, giving the example. It is a figure which shows an example at the time of making the oligonucleotide probe of an indication of this specification into a molecular beacon type probe. It is a figure which shows the absorption spectrum of various oligonucleotides provided with thiazole orange. It is a figure which shows the fluorescence spectrum of various oligonucleotides provided with thiazole orange. It is a figure which shows the fluorescence spectrum of oligonucleotide provided with perylene.

  The present disclosure relates to a fluorescent oligonucleotide probe, and further relates to an immobilized body of the probe and a method for detecting a target nucleic acid using the probe. According to the oligonucleotide probe disclosed herein, in the absence of a target nucleic acid, one fluorophore and two quenchers form a stable stack structure in the stem to increase thermal stability and stability. Quench the light. On the other hand, when a target nucleic acid is present, the oligonucleotide probe hybridizes with the target nucleic acid in a nucleotide chain constituting a base sequence that specifically hybridizes with the target nucleic acid (hereinafter also referred to as probe sequence). . As a result, the stem collapses, and the fluorophore is not quenched by the quencher and emits fluorescence.

  According to the oligonucleotide probes disclosed herein, the stem is thermally stabilized by a fluorophore and a quencher, which makes it easier to obtain a higher melting temperature and a longer or more diverse target nucleic acid ( Probe sequence) can be selected. For this reason, the oligonucleotide probe excellent in the versatility which can design an oligonucleotide probe with a high freedom degree according to the kind of target nucleic acid and hybridization conditions is provided. In addition, by combining two quenchers with one fluorophore, the target nucleic acid can be detected with higher sensitivity by reducing the background level.

  In addition, it is not necessary to place the fluorophore and quencher at both ends like conventional molecular beacons, so it is easy to immobilize on a solid support, avoiding quenching and fluorescence effects due to immobilization. Or it can be suppressed.

  Hereinafter, as an embodiment of the means disclosed in this specification, a fluorescent oligonucleotide probe, an immobilized body thereof, and a method for detecting a target nucleic acid will be described with reference to the drawings as appropriate. FIG. 1 is a diagram showing the action of a molecular beacon probe as an example of the fluorescent oligonucleotide probe disclosed in the present specification, and FIG. 2 is an example of a state in which a molecular beacon probe is provided on a solid phase carrier. FIG.

  In the present specification, the oligonucleotide may have a natural or non-natural base, nucleotide or nucleoside so that it can function to hybridize with the natural, naturally-derived and non-natural DNA or RNA to be detected. That's fine. In addition, the oligonucleotide means about 10 mer to 100 mer in consideration of the use as a probe, but the number is not particularly limited, and even if it exceeds 100 mer, it is included in the oligonucleotide probe in this specification. It is. In addition, the target nucleic acid in the present specification is not particularly limited to a collection source or the like, and natural, naturally-derived or non-natural DNA to be detected (whether single-stranded or double-stranded). Or RNA (which may be single-stranded or double-stranded), or a chimera or hybrid thereof. Naturally-derived DNA means DNA produced by genetic engineering, enzymatic synthesis or chemical synthesis based on the base sequence of natural DNA.

(Oligonucleotide probe)
As shown in FIGS. 1 and 2, the oligonucleotide probe disclosed in the present specification can form a stem and a loop, and includes a fluorophore and a quencher in the stem.

  The oligonucleotide probe disclosed in the present specification may be capable of forming at least one stem and at least one loop, and may be capable of forming a plurality of each. Therefore, as shown in FIG. 1 and FIG. 2, it may be possible to form a so-called molecular beacon type probe having one stem and one loop, or a plurality of tRNAs or similar structures. And a plurality of loops may be formed. As will be described later, the oligonucleotide probe disclosed in the present specification can obtain sufficient detection specificity even by adopting the simplest molecular beacon type.

  In addition, the oligonucleotide probe disclosed in the present specification is capable of forming a stem and a loop, and includes a pair of stem-forming strands that pair with each other to form at least one stem. It means that the stem to be formed has a loop-forming chain that continuously forms a loop. In this specification, when only referring to a stem or loop, it is intended that these are formed.

  Although the length of the stem-forming chain | strand which forms a stem is not specifically limited, Each stem-forming chain | strand can be made into the chain length which forms 4 or more and 9 or less base pairs. The molecular beacon type probe preferably has a chain length that forms 8 to 40 base pairs, more preferably 15 to 30. In addition, the stem in the oligonucleotide probe disclosed in the present specification is not limited to one configured on both ends of the oligonucleotide chain. One stem-forming strand may be at a portion other than the end of the oligonucleotide probe and the other stem-forming strand may be at the probe end, or both stem-forming strands may be at portions other than the probe end.

  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.

  The oligonucleotide probes disclosed herein comprise at least one fluorophore on one stem-forming strand of at least one stem and at least two quenchers on the other stem-forming strand, On the other hand, two quenchers are arranged to be pairable. That is, a pairing unit composed of one fluorophore and two fluorophores can be formed in the stem. The number and arrangement form of the pairing units are not particularly limited. As shown in FIG. 1, the number of pairing units may be one in the stem, or two or more. In addition, as shown in FIG. 1, for example, the pairing unit includes one fluorophore in one stem-forming strand and two quenchers in the other stem-forming strand to form a pairing unit. In addition to this pairing unit, one stem-forming strand is equipped with multiple fluorophores, and the other stem-forming strand is equipped with multiple quenchers (twice multiple fluorophores) to match the number of fluorophores A unit may be formed. Furthermore, one stem-forming strand may be provided with a fluorophore of one pairing unit, and a quencher of another pairing unit may be provided.

  By making such a pairing unit formable, it is sensitive even at the position in the stem that is not on the end of the probe but on the inner side of the probe from the 5 'or 3' end to the 3 base equivalent site or the 4 base equivalent site. The target sequence can be detected well. That is, even in the oligonucleotide sequence, dependence on the sequence can be suppressed. Even when the target sequence extends from the loop to the stem, the target sequence can be detected with high sensitivity.

  When a plurality of pairing units can be formed in one stem, different types of fluorophores can be used, but preferably a combination of fluorophores that emit fluorescence that can be detected in the same wavelength range and quench to the same extent. Is preferably used. More preferably, the same fluorophore is used. Further, as long as two quenchers can be quenched in one pairing unit, they may be the same or different but may be the same.

  Two quenchers constituting one pairing unit are continuously provided on one stem-forming strand. That is, no nucleotide is included between the two quenchers. If multiple pairing units can be formed, one or more nucleotides may or may not be present between the fluorophores on the same stem-forming strand of adjacent pairing units. In the case of quenching due to the association, it is preferable that there are one or more nucleotides. Similarly, there may or may not be one or more nucleotides between quenchers on the same stem-forming strand of adjacent pairing units. The base in the intervening nucleotide is not particularly limited, and may be selected from purine bases and / or pyrimidine bases, or may be an artificially synthesized base.

  Further, in the paired stem-forming strands, neither the fluorophore nor the quencher is linked outside the 5 'end and 3' end of the oligonucleotide probe. Even when the fluorophore and quencher are located at the most terminal side, they are located inside the most terminal nucleotide (more 3 'when the most terminal is the 5' end, more when the 3 'end is the same). At the 5 ′ end).

  The fluorophore is located in the stem as described above, but is preferably located within the probe sequence of the oligonucleotide probe. This is because, when the fluorophore forms a double strand by hybridization with a target nucleic acid depending on the type, the fluorescence may be enhanced by intercalation within the double strand. Such a fluorophore will be described later.

  The fluorophore and quencher are also not particularly limited in the type of bases of adjacent nucleotides and in the base type of distal nucleotides adjacent to the nucleotides.

  The fluorophore is contained as X in the unit represented by the formula (1) between the nucleotides of the stem-forming strand. The fluorophore (X) is connected to a unit part 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 fluorophore 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 fluorophore and quencher paired in the stem, the carbon number of the alkylene chain in R 1 in formula (1) and formula (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 fluorophore. Examples of the fluorophore 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 fluorophore may be appropriately derivatized in order to be linked to the unit represented by the formula (1), or may be substituted between the linking group in consideration of pairing with the quencher. An alkylene chain, an alkenylene group or an alkynylene group which may be formed may be added. Moreover, the atom on the fluorophore connected with respect to the unit represented by Formula (1) is not specifically limited.

  Examples of fluorophores whose fluorescence is enhanced by intercalation include thiazole orange, oxazole orange, and perylene. These fluorophores are preferably disposed within the probe sequence and intercalated. On the other hand, if the fluorescence intensity of the fluorophore is reduced by intercalation due to the combination of fluorophore, R1, and R2, the beacon is designed so that the fluorophore does not intercalate and conversely the quencher intercalates. Is preferred.

  Examples of the fluorophore (X) linked in the 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 quencher is contained in the unit represented by the following formula (2) between the nucleotides of the stem-forming strand. That is, the quencher (Y) is connected to a unit part other than Y as a part of the unit, and is consequently 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 quencher. The quencher is not particularly limited as long as it can quench the fluorophore used 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 quencher is preferably any one selected from methyl red, azobenzene, and methylthioazobenzene. A typical azo quencher is a quencher of a type in which the HOMO of the fluorophore is lower than the HOMO of the quencher in relation to a polycyclic condensed fluorophore such as perylene, in other words, a quencher having a strong reducing power. It can be said.

  Further, examples of the quencher 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 an LUMO lower than that of a fluorophore can be used. However, an electron-withdrawing substituent such as a nitro group or a carboxyl group that does not raise the LUMO energy level. Is preferred. Anthraquinone is preferred. Typical anthraquinone quenchers such as anthraquinone are quenchers that satisfy the condition that the LUMO of the fluorophore is higher than the LUMO of the quencher in relation to the fluorophore of the condensed aromatic ring system such as perylene. It can be called char.

In addition, from the viewpoint of quenching ability, the quencher preferably has a maximum absorption wavelength within 150 nm that is different from the maximum absorption wavelength of the fluorophore forming the pair. The difference between the maximum absorption wavelength of the quencher 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 quencher is on the shorter wavelength side than the maximum absorption wavelength of the fluorophore. In relation to the fluorophores used, by controlling the maximum absorption wave length of the quencher, it is possible to optimize the quencher.

  Control of the maximum absorption wavelength of the quencher (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 quencher. . 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 quencher may be appropriately derivatized to be linked to the unit represented by the formula (2), or substituted between the linking groups in consideration of pairing with the quencher. An alkylene chain which may be used may be added. Moreover, the connection part with the connection group of a quencher is selected suitably.

  Examples of the quencher (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 fluorophore connected with respect to the unit represented by Formula (2) is not specifically limited.

  The combination of the fluorophore and the quencher disclosed in this specification can be selected based on the fluorescence quenching action shown below. In general, there are two types of fluorescence quenching: 1) Forster type excitation energy transfer and 2) Electron transfer type. In fluorescence quenching based on Forster-type energy transfer, the fluorescence spectrum of the fluorophore and the absorption spectrum of the quencher need to overlap. Therefore, when using Förster-type excitation energy transfer, it is preferable to select a combination of a fluorophore and a quencher that satisfies these conditions. In the case of the Forster type, quenching can be performed even if the distance between the fluorophore and the quencher is 2 to 10 nm, but in the present disclosure, the fluorophore and the quencher are in contact with each other. As long as it is satisfied, it can be sufficiently quenched with the Förster type.

  On the other hand, in the electron transfer type 2), the relationship between the energy ranks of the highest occupied orbit (HOMO) and the lowest unoccupied orbit (LUMO) of the fluorophore and the quencher is important. The fluorescence mechanism of fluorophore is based on the emission of excitation energy in the form of fluorescence when electrons excited from HOMO to LUMO return to HOMO again. When one of the two electrons in the HOMO of the fluorophore is photoexcited and moves to LUMO, the HOMO of the fluorophore becomes empty. When the energy level of the quencher HOMO is higher than the HOMO of the fluorophore, electrons in the quencher HOMO move to the HOMO of the fluorophore that has become empty. Then, the excited fluorophore electrons cannot return to the original HOMO, and therefore cannot emit fluorescence. In addition, when the LUMO of the quencher is lower than the HOMO of the fluorophore, electrons that are excited and transition to the LUMO move to the LUMO of the quencher, and therefore cannot return to the original HOMO and cannot emit fluorescence. That is, the electron quenching type fluorescence quenching is preferably a combination that satisfies the conditions that 1) the HOMO of the fluorophore is lower than the HOMO of the quencher, or 2) the LUMO of the fluorophore is higher than the LUMO of the quencher. Furthermore, since the electron transfer occurs only at a short distance, it is desirable that the fluorophore and the quencher are in contact. In the present disclosure, since the fluorophore and the quencher are in contact (stacking), a combination satisfying even the above conditions can be sufficiently quenched by the electron transfer type. Note that most of the combinations of the fluorophore and the quencher disclosed in this specification satisfy the conditions for causing electron transfer type quenching.

  In addition, quenching of a condensed aromatic ring system fluorophore such as perylene by anthraquinone or a derivative thereof occurs when excited electrons move to LUMO of anthraquinone. That is, quenching by “electron trap”. In addition to the fact that the electron transfer type quench phenomenon is inherently easy to move electrons, the electron transfer is further promoted by the strong pairing between the anthraquinone quencher and the condensed aromatic ring fluorophore. Is inferred. As the number of pairs of anthraquinone quenchers and fused aromatic ring fluorophores increases, this electron transfer trapping phenomenon is further enhanced, and the pair of quenchers that do not form pairs from the fluorophores. It is thought that it is further strengthened by the fact that electrons are easily transferred to char through natural base pairs. On the other hand, the azo-type quencher is a “hole trap” type that is a quench caused by the transfer of electrons present in the HOMO of the azo-type quencher to holes generated in the HOMO by excitation of the fluorophore. . Since electrons have a higher mobility than holes, the “electron trap” type perylene-anthraquinone can be more efficiently quenched.

  Introduction of a fluorophore and a quencher can be achieved, for example, by using an amidite derivative that introduces this type of unit in place of an amidite derivative corresponding to a 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 fluorophore or quencher 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 fluorophore or quencher 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 with linked char can be synthesized.

  In addition, the fluorophore or quencher 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 fluorophore or a quencher in which a carboxylic acid group or an isocyanate group is introduced or retained so as to be reactive with the amino group. Fluorophore or the like may be introduced by reacting char.

  Since the oligonucleotide probe disclosed in the present specification described above is a probe that self-generates a fluorescent signal, it is not necessary to label the target nucleic acid with a fluorescent substance or the like, and the design flexibility of the target nucleic acid is high. There is an advantage that detection with high specificity is possible. Furthermore, since the fluorophore and the quencher are provided in the stem, it is possible to avoid or suppress the influence of the immobilization on the light emission and quenching as well as the ease of immobilization on the solid phase carrier.

  Oligonucleotide probes disclosed in the present specification include detection of target nucleic acids in a liquid phase such as real-time PCR to which molecular beacons are conventionally applied, gene expression patterns, disease classification, analysis of causative genes of diseases, genetic diagnosis, etc. It is advantageous for gene expression monitoring, detection of polymorphisms such as SNP, and detection of various mutations. In particular, it is advantageous for detection of polymorphisms and mutations, and is further advantageous for detection of comprehensive polymorphisms and mutations. In particular, a stem provided with the fluorophore and quencher of the oligonucleotide probe disclosed in the present specification has higher thermal stability and an increase in Tm than the same stem except that the fluorophore and quencher are not included. Therefore, a probe sequence having a long sequence as a target nucleic acid can be provided, and highly specific DNA analysis can be performed. In addition, in an array in which a molecular beacon probe capable of simultaneously detecting a plurality of target nucleic acids having a large number of SNPs or mutation sites, preferably several dozens or more, is immobilized, it is sufficient to distinguish the target nucleic acids. It is necessary to have a long probe sequence. In the case of a conventional molecular beacon, when the stem is lengthened and the loop is in such a case, the oligonucleotide probe disclosed in this specification can have a probe sequence in a range in which the Tm of the stem is high and the fluorophore is included. As a result, it is advantageous in that the probe sequence can be secured longer.

(Probe immobilized body)
The probe-immobilized body disclosed in the present specification can comprise a solid phase carrier and an oligonucleotide probe disclosed in the present specification, which has a base sequence that can specifically hybridize with the target nucleic acid. . In the probe-immobilized body disclosed in the present specification, it is preferable to use a molecular beacon as a probe.

  The solid phase carrier may be, for example, a bead or a flat plate, and the material is not particularly limited, but a solid phase carrier made of glass or plastic can be used. Preferably, the solid support is a flat plate and is an array in which two or more kinds of oligonucleotide probes are fixed in a fixed sequence. The array can fix a large number of oligonucleotide probes, and is convenient for comprehensively detecting various polymorphisms and mutations. In addition, the array may include a plurality of partitioned individual array regions on a single solid phase carrier. In these individual array regions, the same set of oligonucleotide probes may be immobilized, or different sets of oligonucleotide probes may be immobilized.

  The immobilization form of the oligonucleotide probe is not particularly limited. It may be covalent or non-covalent. The oligonucleotide probe can be immobilized on the surface of the solid phase carrier by various conventionally known methods. An appropriate linker sequence may be provided on the surface of the solid phase carrier. Since the oligonucleotide probe disclosed in the present specification does not have a fluorophore and a quencher at its end, it can be easily immobilized on a solid phase carrier by the same method as in the prior art.

  The use of such a probe-immobilized body is not particularly limited, and is advantageous for gene expression patterns, disease classification, analysis of causative genes such as diseases, gene expression monitoring such as gene diagnosis, detection of polymorphism such as SNP, and detection of various mutations. It is. In particular, it is advantageous for detection of polymorphisms and mutations, and is further advantageous for detection of comprehensive polymorphisms and mutations.

(Target nucleic acid detection method)
The method for detecting a target nucleic acid disclosed in the present specification uses the oligonucleotide probe disclosed in the present specification, which has a base sequence capable of specifically hybridizing with the target nucleic acid, and the molecular beacon. The signal based on the fluorophore can be detected. In the detection method disclosed in the present specification, the oligonucleotide probe is preferably a molecular beacon, more preferably, a plurality of oligonucleotide probes (typically molecular beacons) immobilized on a solid phase carrier. It is preferable to use a body.

  The detection method disclosed in the present specification can be performed, for example, as follows. First, an array is prepared in which molecular beacons that can specifically hybridize with a plurality of target nucleic acids are immobilized on a solid phase carrier. After that, a nucleic acid sample that may contain the target nucleic acid (typically, one obtained by extracting nucleic acid from a biological sample collected from blood or tissue or further amplified by PCR or the like under certain conditions) The molecular beacon is supplied to the molecular beacon on the array in the presence of a hybridization solution of a predetermined composition. A hybridization reaction is carried out under predetermined hybridization conditions. The hybridization temperature is preferably lower than the Tm of the stem and the Tm of the probe sequence and the target nucleic acid. After elapse of a predetermined time, the array is washed, and hybridization with the target nucleic acid present in the nucleic acid sample is performed by detecting a fluorescent signal emitted from the fluorophore. The fluorescence signal can be detected by a fluorescence scanner or the like. Based on the acquired fluorescence signal, analysis necessary for detection, identification, quantification, etc. of the target nucleic acid is performed.

  Hereinafter, the disclosure of the present specification will be specifically described by way of examples, but the following examples are not intended to limit the disclosure to the present specification.

(Amidite monomerization of D-threoninol protected with an allyloxycarbonyl group)
In this example, an amidite monomer (compound A) having a unit (carbon number 3) linking a fluorophore or a quencher was synthesized according to the following scheme. D-threoninol (0.99 g, 9.41 mmol) was dissolved in 75 ml of tetrahydrofuran (THF) in a 300 ml eggplant flask, and 15 ml of triethylamine was added and stirred. Next, allyl chloroformate (1.01 ml, 9.51 mmol) dissolved in 75 ml of THF was added dropwise to the above THF solution in an ice bath. After 15 minutes, the ice bath was removed and the temperature was returned to room temperature. Stirring was continued as it was, and the reaction was stopped 1 hour and 30 minutes after the dropwise addition of the THF solution. Then, the solvent was distilled off with an evaporator and purified by silica gel column chromatography (developing solvent: chloroform: methanol = 3: 1) to obtain Compound 1-1.

  Next, the obtained compound 1 (1.72 g, 9.09 mmol) was collected in a 200 ml two-necked eggplant flask and dissolved by adding 30 ml of dehydrated pyridine under nitrogen, and N, N, diisopropylethylamine was added thereto. (DIPEA: 1.54 mL, 9.09 mmol) was added and stirred. Next, dimethoxytrityl chloride (DMT-Cl: 3.08 g, 9.09 mmol) and dimethylaminopyridine (DMAP: 0.14 g, 1.14 mmol) were added to a 50 ml two-necked eggplant flask, and further 10 ml of dehydrated dichloromethane as a solvent. Was added and dissolved. This dichloromethane solution was slowly added dropwise to the above pyridine solution in an ice bath. After stirring in an ice bath for about 15 minutes, the ice bath was removed and stirring was continued at room temperature, and the reaction was stopped 4 hours and 30 minutes after the dropwise addition of the dichloromethane solution. After the solvent was distilled off with an evaporator, the residue was purified by a silica gel column chromatography method (developing solvent hexane: ethyl acetate: triethylamine = 66: 33: 3) to obtain Compound 1-2.

  Compound 1-2 (0.74 g, 1.51 mmol) was collected in a two-necked eggplant flask and azeotroped with 8 ml of dehydrated acetonitrile three times to remove moisture, and then 30 ml of dehydrated acetonitrile was added and dissolved. 2-Cyanoethyl N, N, N ′, N′-tetraisopropyl phosphorodiamidite (2-cyanethyl N, N, N ¢, N ¢ -tetraisopropylphosphodiamidite: 0.54 g, 1.79 mmol) was added and stirred. Collect 1H-tetrazole (1H-tetrazole: 0.137 g, 1.51 mmol) in another two-necked eggplant flask, remove water by azeotropic distillation with 8 ml of dehydrated acetonitrile three times, and dissolve by adding 15 ml of dehydrated acetonitrile. I let you. The 1H-tetrazole solution was added dropwise to the acetonitrile solution of the above compound 1-2 in an ice bath, stirred for about 15 minutes, the ice bath was removed, and the mixture was returned to room temperature and further stirred. Thereafter, the reaction was stopped in about 1 hour 30 minutes. Thereafter, the solvent was distilled away with an evaporator, and the remaining oily compound was dissolved by adding ethyl acetate. The ethyl acetate solution was shaken twice with a saturated aqueous solution of sodium bicarbonate using a separatory funnel, followed by two similar shakes with a saturated aqueous solution of sodium chloride. Thereafter, magnesium sulfate was added to remove water, and then ethyl acetate was distilled off with an evaporator. Purification by silica gel column chromatography (developing solvent hexane: ethyl acetate: triethylamine = 50: 50: 3) gave compound A. It was.

(Amidite monomerization of 3-amino-1,2-propanediol protected with an allyloxycarbonyl group)
In this example, an amidite monomer (compound B) having a unit (carbon number 2) linking a fluorophore or a quencher was synthesized according to the following scheme.

  3-amino 1,2-propanediol (1.0 g, 11 mmol) was dissolved in 30 ml of dimethylformamide (DMF) in a 200 ml eggplant flask, and 15 ml of triethylamine was added and stirred. . Next, allyl chloroformate (1.4 ml, 11 mmol) dissolved in 20 ml of DMF was added dropwise to the DMF solution in an ice bath. After 15 minutes, the ice bath was removed and the temperature was returned to room temperature. Stirring was continued as it was, and the reaction was stopped 2 hours after the DMF solution was added dropwise. Then, the solvent was distilled off with an evaporator and purified by silica gel column chromatography (developing solvent: chloroform: methanol = 3: 1) to obtain compound 1-3. Compound 1-4 and then Compound B were obtained in the same manner as in Example 1 except that Compound 1-3 was used as a raw material.

  Compound C shown below is a method described in Nature Protocols, Vol. 2, pages 203 to 212, and Compound D is Journal of the American Society (Journal of the American Chemical Society). Compound E was obtained by the method described in Chemical Society, 2003, 125, 2217-2223, and by the method described in Tetrahedron Letters, 2007, 48, 6759-6762.

(Synthesis of thiazole orange (TO-1, TO-2))
In this example, a thiazole orange derivative as a fluorophore was synthesized according to the following scheme.

  2- (Methylthio) benzothiazole (2.04 g, 11.3 mmol) is weighed into a 200 ml eggplant flask, dissolved by adding 5 ml of ethanol, and iodomethane (1.5 ml, 24.1 mmol) is added to this solution. Then, the mixture was heated to reflux at 65 ° C. for 3 hours. The produced precipitate was collected by suction filtration to obtain Compound 2-1 (0.52 g).

  Next, bromoacetic acid (1.94 g, 14.0 mmol) was weighed into a 200 ml eggplant flask and dissolved by adding 10 mL of ethyl acetate. To this was added lepidin (2 ml, 15.1 mmol) and stirred at room temperature for 2 hours 30 minutes. The produced precipitate was collected by suction filtration to obtain Compound 2-2 (0.41 g). In the same manner as described above, bromobutyric acid (8.08 g, 52.8 mmol) was then weighed into a 200 ml eggplant flask, and 10 ml of ethyl acetate was added and dissolved. To this was added lepidin (7 ml, 52.8 mmol) and stirred at 80 ° C. overnight. The produced precipitate was collected by suction filtration to obtain Compound 2-3 (4.85 g).

  Thiazole Orange TO-1 was prepared by dissolving Compound 2-1 (0.42 g, 2 mmol) and Compound 2-2 (0.41 g, 2 mmol) by adding 10 ml of dehydrated ethanol, refluxing at 60 ° C. for 5 minutes, and then triethylamine ( 0.27 ml, 2 mmol) was added, and after turning red, the mixture was returned to room temperature with stirring. A precipitate formed by adding 150 ml of diethyl ether to the red reaction solution was collected by suction filtration to obtain thiazole orange (TO-1, 0.59 g). Thiazole orange TO-2 was obtained in the same manner except that compound 2-3 was used.

(Synthesis of oligonucleotides)
In this example, oligonucleotides introduced with the following quenchers and oligonucleotides introduced with fluorophores were synthesized. However, PE represents perylene, MR represents methyl red, and Azo represents azobenzene.

(Synthesis of oligonucleotides incorporating azobenzene, methyl red, and perylene)
For synthesis of DNA with azobenzene, methyl red, or perylene as quencher, use ABI394 type DNA synthesizer, corresponding to phosphoramidite monomer (compound C, D, E) and four natural bases Using the commercially available phosphoramidite monomer, Nature Protocols, 2007, Vol. 2, pages 203 to 212, was synthesized and purified.

(Introduction of thiazole orange (TO-1, TO-2) and FITC into oligonucleotide)
Thiazole orange (TO-1, TO-2) and FITC were introduced into the oligonucleotide by the method shown in the following scheme via a phosphoramidite monomer protected with an allyloxycarbonyl group.

Phosphoramidite monomer A in which amino group of D-threoninol is protected with allyloxycarbonyl group using ABI394 type DNA synthesizer A, commercially available phosphoramidite monomer corresponding to four natural bases, and amidite monomer described above Using C, D, and E as appropriate, DNA having a predetermined sequence was extended on a controlled pore glass (CPG) carrier (Compound 3-1). The CPG carrier (10 mg, 0.45 μmol) was weighed in a plastic syringe equipped with a filter, and washed 3 times with 1 mL of acetonitrile, and then 3 times with 1 mL of dichloromethane. Next, 48.8 μL (450 μmol) of N-methylaniline was added to 500 μL of a dichloromethane solution in which Pd (Ph ₃ ) ₄ (5.2 mg, 4.5 μmol) was dissolved, and this was added to the above-mentioned CPG carrier (compound 4-1). In addition to the above, by reacting at 35 ° C. for 3 hours, only the allyloxycarbonyl group was deprotected from the CPG carrier (Compound 3-2).

  Next, thiazole orange TO-1 (10 mg, 45 μmol) and pyridinium para-toluenesulfate (PPTS: 11.3 mg, 45 μmol) were dissolved in 400 μL of DMF, and benzotriazol-1-yl-oxyphosphorylphosphonium 59 μm: ) To which 100 μl of DMF solution was added was added to the syringe containing the CPG carrier (Compound 3-2) and shaken at room temperature for 3 days. After this, the reaction solution was removed by filtration, and then 1 mL of 0.1 M PPTS in DMF was added to the syringe and shaken for 1 minute to wash the CPG carrier, and then 3 times with 1 mL of DMF, followed by 3 mL with 1 mL of dichloromethane. The CPG carrier into which TO-1 was introduced was obtained by washing twice (compound 3-3).

  Subsequently, DNA was excised from the CPG carrier by the method described in Nature Protocols 2007, Volume 2, pages 203 to 212, and the target was obtained by high performance liquid chromatography using the method described in the same page. DNA was separated and purified (Compound 3-4).

  DNA into which thiazole orange TO-2 was introduced was obtained by the same method as described above except that TO-2 was used instead of TO-1.

  FITC-introduced DNA was prepared by adding DIPEA (6.12 μl, 18 μmol) to a DMF solution (500 μL) of FITC (14.02 mg, 18 μmol), and adding this to a CPG carrier (10 mg, 0.36 μmol) in which only the allyloxycarbonyl group was deprotected. ) In addition to (compound 3-2), the mixture was stirred for 3 days, and then prepared by the same operation as that for TO-introduced DNA.

  Except for using compound B instead of compound A, TO-1, TO-2, or FITC is introduced to synthesize an oligonucleotide containing a linking group R1 (C2: 2 carbon atoms) in the same manner as described above. (Compound 3-5).

(Confirmation of fluorescence quenching by combination with quencher)
In order to confirm that various fluorophores were quenched by “base pairing” with the quencher, fluorescence and absorption spectra were measured using the oligonucleotide synthesized in Example 4. The conditions of the solution for measuring the absorption spectrum and the fluorescence spectrum are as follows.

Fa sequence oligonucleotide: 5 μmol / l
Qb sequence or Nb oligonucleotide: 5 μmol / l
Sodium chloride: 0.1 mol / l
Phosphate buffer: 10 mmol / l (pH 7.0)

  In addition, as a target nucleic acid of various oligonucleotides Fa containing a fluorophore, an oligonucleotide (without quencher) (Nb) having a sequence complementary to Fa was separately prepared. The results are shown in FIGS. 3A and 3B.

  FIG. 3A shows an absorption spectrum at 20 ° C. of each of Fa-C2-TO1 (single strand) and Fa-C3-TO1 / Qb-MR duplex, and FIG. 3B shows a fluorescence spectrum. As shown in FIG. 3A, the absorption at 515 nm based on thiazole orange was greatly reduced by duplex formation with Qb-MR. This fact indicates that the quencher methyl red and thiazole orange are stacked. In addition, the melting temperature Tm of the Fa-C2-TO1 / Qb-MR duplex is 54 ° C., and a natural DNA duplex (5′-GGTATCGCAATC-3 ′ / 3′-CCATAGCGTTTAG not containing thiazole orange and methyl red). -5 ′) higher than Tm (48 ° C.). This fact also supports the strong stacking of thiazole orange and methyl red. Similar changes in absorption spectrum and increase in Tm were also observed in Fa-C2-TO2 / Qb-MR, Fa-C3-TO1 / Qb-MR, and Fa-C3-TO2 / Qb-MR.

  As shown in FIG. 3B (excitation at 511 nm), the fluorescence intensity at 533 nm based on thiazole orange from the Fa-C2-TO1 / Qb-MR duplex is compared to Fa-C2-TO1 in the single-stranded state. It decreased to 1/10. On the other hand, when Fa-C2-TO1 was duplexed with a complementary oligonucleotide (Nb), the fluorescence intensity at 533 nm was doubled at 20 ° C. compared to Fa-C2-TO1 in a single-stranded state. Therefore, it was found that when a beacon is created with this combination, a signal / noise ratio of about 20 times can be expected.

  Next, Fa-C3-PE containing perylene was duplexed with Qb-MR or Qb-Azo. The results are shown in FIG. As shown in FIG. 4, the fluorescence intensity at 461 nm when excited at 425 nm is 1/15 in Qb-MR and 20% at 20 ° C. compared to single-chain Fa-C3-PE. -Azo decreased to 1/30. On the other hand, when Fa-C3-PE was double-stranded with Nb, fluorescence comparable to that of single-chain Fa-C3-PE was observed. Therefore, it was found that a signal / noise ratio of about 30 times around 20 ° C. can be expected when perylene is combined with azobenzene.

  Based on the results of Example 5, molecular beacon-type oligonucleotide probes shown in Table 2 below were designed and synthesized. Various molecular beacons were synthesized according to the method described in Example 4 using the various amidide monomers and various fluorophores and quenchers synthesized or prepared in Examples 1-3. Various sequences are also shown.

* The underlined part is the stem-forming chain.
Note that the FITC at the 5 ′ end and the MR at the 3 ′ end used in MB0F of the comparative example have the following structures, unlike the linker used for introduction into the beacon disclosed in this specification.

  Among these beacons, changes in fluorescence intensity were observed before and after MBt as a target nucleic acid was added to MB1-C2-TO1-MR and MB2-C2-TO1-MR. The conditions of the solution for measuring the fluorescence spectrum were as follows. The results are shown in Tables 3 and 4.

Concentration of beacon: 0.2 μmol / l
Concentration of target nucleic acid Nb: 0.4 μmol / l
Sodium chloride: 0.1 mol / l
Phosphate buffer: 10 mmol / l (pH 7.0)

<MB1-C2-TO1-MR>
The fluorescence intensity from 531 nm thiazole orange upon excitation at 520 nm before and after adding MB-t to MB1-C2-TO1-MR is shown in the table below.

(Fluorescence measurement conditions) Spectrofluorometer: FP-6500 manufactured by JASCO; excitation bandwidth: 3 nm; fluorescence bandwidth: 3 nm; response: 0.1 sec; sensitivity: high; scanning speed: 200 nm / min

  As shown in Table 3, this molecular beacon type probe had a stable background value (before addition of MB-t) over a wide temperature range and a stable signal intensity ratio at a wide temperature. From comparison with a comparative example described later, it was found that this molecular beacon probe tends to be excellent in thermal stability of the stem. Moreover, since the signal intensity ratio was also stabilized, it turned out that it is convenient for detecting mutations, such as SNP and a mutation.

<MB2-C2-TO1-MR>
The fluorescence intensity from 531 nm thiazole orange upon excitation at 510 nm before and after adding MBt to MB2-C2-TO1-MR is shown below.

(Fluorescence measurement conditions) Spectrofluorometer: FP-6500 manufactured by JASCO; excitation bandwidth: 3 nm; fluorescence bandwidth: 3 nm; response: 0.1 sec; sensitivity: high; scanning speed: 200 nm / min

  As shown in Table 4, the molecular beacon type probe had a stable background (before MB-t addition) from 30 ° C. to 50 ° C., and the signal intensity ratio tended to stabilize in this range. From comparison with a comparative example described later, it was found that this molecular beacon probe tends to be excellent in thermal stability of the stem. Moreover, since the signal intensity ratio was also stabilized, it turned out that it is convenient for detecting mutations, such as SNP and a mutation.

<Comparative example: MB0F>
As a comparative example, the fluorescence intensity from 520 nm FITC in excitation at 494 nm before and after adding MBt to MB-cont is shown below.

(Conditions for fluorescence measurement) Spectrofluorometer: FP-6500 manufactured by JASCO; excitation bandwidth: 3 nm; fluorescence bandwidth: 3 nm; response: 0.1 sec; sensitivity: middle; scanning speed: 200 nm / min

  As shown in Table 5, in the conventional molecular beacon, the bag ground (before MB-t addition) increased with a temperature rise, and increased rapidly at 40 ° C. or higher. On the other hand, luminescence due to hybridization (after addition of MB-t) rapidly decreased at 50 ° C. or higher. As a result, at 40 ° C. or higher, the signal intensity ratio suddenly decreased. Since relatively high hybridization temperatures are required to detect SNPs and mutations, it was found that they are not suitable for detection of SNPs and mutations. Further, from the instability, it was estimated that there was more variation in the solid phase reaction, and it was found that the solid phase reaction is not suitable.

<MB1-C3-PE-Azo>
The fluorescence intensity from 460 nm perylene in the excitation at 425 nm before and after adding MBt to MB1-C3-PE-Azo is shown in the following table.

(Conditions for fluorescence measurement) Spectrofluorometer: FP-6500 manufactured by JASCO; excitation bandwidth: 3 nm; fluorescence bandwidth: 3 nm; response: 0.1 sec; sensitivity: middle; scanning speed: 200 nm / min

<MB2-C3-PE-Azo>
The following table shows the fluorescence intensity from 460 nm perylene in the excitation at 425 nm before and after adding MBt to MB2-C3-PE-Azo.

(Conditions for fluorescence measurement) Spectrofluorometer: FP-6500 manufactured by JASCO; excitation bandwidth: 3 nm; fluorescence bandwidth: 3 nm; response: 0.1 sec; sensitivity: middle; scanning speed: 200 nm / min

  As shown in Tables 6 and 7, it was found that the change in the fluorescence intensity ratio due to the difference in hybridization temperature was suppressed.

The compound Anth shown below was obtained by the method described in Chemical Communications, Vol. 2006, 5062-5064.

(Synthesis of amidite monomers containing 4- [4-methylthiophenylazo] benzoic acid)
According to the following scheme, an amidite monomer containing 4- [4-methylthiophenylazo] benzoic acid was synthesized. First, ethyl para-nitrosobenzoate (4-1) was synthesized by the method described in Journal of the Organic Chemistry, 1970, Vol. 35, pages 505-508. This nitroso compound 4-1 was placed in a two-necked eggplant flask and purged with nitrogen, p-methylthioaniline 4-2 was added, acetic acid was further added as a solvent, and the reaction was allowed to proceed overnight under light shielding. Distilled water was added to the reaction solution, ethyl acetate was added, and the mixture was sufficiently shaken with a separatory funnel. Subsequently, the ethyl acetate layer was washed successively with distilled water, a saturated aqueous sodium bicarbonate solution, and a saturated aqueous sodium chloride solution and dried over magnesium sulfate, and then the ethyl acetate as a solvent was distilled off. The resulting product was further purified by silica gel chromatography (hexane: ethyl acetate = 15: 1) to give compound 4-3 (yield 50%).

  Ethanol and 2N aqueous sodium hydroxide solution were added to the obtained compound 4-3 for dissolution, and the mixture was stirred for 17 hours while being shielded from light. After that, hydrochloric acid was added to adjust the pH to about 5, and then ethyl acetate was added, followed by sufficient shaking with a separatory funnel. Subsequently, the ethyl acetate layer was washed successively with distilled water, saturated aqueous sodium bicarbonate solution and saturated aqueous sodium chloride solution and dried over magnesium sulfate, and then the ethyl acetate solvent was distilled off to give para-nitrosobenzoic acid 4-4. (Yield 99% or more). Next, amidite monomer 4-5 was prepared according to the method described in Nature Protocols, Vol. 2, pages 203 to 212 except that para-nitrosobenzoic acid 4-4 was used instead of azobenzene. Obtained.

(Synthesis of amidite monomer containing Cy3)
Amidite monomers containing Cy3 were synthesized according to the following scheme. First, Cy3 (5-1) was obtained by the method described in Journal of the American Chemical Society 2001, Vol. 125, pages 361-362. Next, amidite monomer 5-2 was obtained according to the method described in Nature Protocols, Vol. 2, pages 203 to 212 except that Cy3 (5-1) was used instead of azobenzene. .

  Subsequently, molecular beacon-type oligonucleotide probes shown in Table 6 below were designed and synthesized. Various molecular beacons were synthesized according to the method described in Example 4 using the various amidide monomers and various fluorophores and quenchers synthesized or prepared in Examples 1-3. Various sequences are also shown.

The abbreviations in Table 8 are as shown in the following figure. The abbreviations used in Table 2 of Example 6 are also exactly the same as those in the following figure.

For these beacons, changes in fluorescence intensity before and after the addition of MBt as the target nucleic acid were observed. The conditions of the solution for measuring the fluorescence spectrum were as follows. The results are shown in Tables 9-15.
Concentration of beacon: 0.2 μmol / l
Concentration of target nucleic acid Nb: 0.4 μmol / l
Sodium chloride: 0.1 mol / l
Phosphate buffer: 10 mmol / l (pH 7.0)

1. Quenching with perylene- (4- [4-methylthiophenylazo] benzoic acid-Confirmation of luminescence <MB1-C3-PE-S>
The fluorescence intensity from 460 nm perylene in the excitation at 425 nm before and after adding MBt to MB1-C3-PE-S is shown in the following table.

(Conditions for fluorescence measurement) Spectrofluorometer: FP-6500 manufactured by JASCO; excitation bandwidth: 3 nm; fluorescence bandwidth: 3 nm; response: 0.1 sec; sensitivity: middle; scanning speed: 200 nm / min

<MB2-C3-PE-S>
The following table shows the fluorescence intensity from 460 nm perylene in the excitation at 425 nm before and after adding MBt to MB2-C3-PE-S.

(Conditions for fluorescence measurement) Spectrofluorometer: FP-6500 manufactured by JASCO; excitation bandwidth: 3 nm; fluorescence bandwidth: 3 nm; response: 0.1 sec; sensitivity: middle; scanning speed: 200 nm / min

  As shown in Tables 9 and 10, by using 4- [4-methylthiophenylazo] benzoic acid for perylene, the quenching-luminescence phenomenon could be confirmed by supplying the target nucleic acid. Therefore, it was found that perylene can be used as a fluorophore and 4- [4-methylthiophenylazo] benzoic acid as a quencher.

2. Confirmation of quenching-luminescence by perylene-anthraquinone <MB1-C3-PE-Q>
The fluorescence intensity from 460 nm perylene in the excitation at 425 nm before and after adding MBt to MB1-C3-PE-Q is shown in the following table.

(Conditions for fluorescence measurement) Spectrofluorometer: FP-6500 manufactured by JASCO; excitation bandwidth: 3 nm; fluorescence bandwidth: 3 nm; response: 0.1 sec; sensitivity: middle; scanning speed: 200 nm / min

<MB2-C3-PE-Q>
The fluorescence intensity from 460 nm perylene in the excitation at 425 nm before and after adding MBt to MB2-C3-PE-Q is shown in the following table.

(Conditions for fluorescence measurement) Spectrofluorometer: FP-6500 manufactured by JASCO; excitation bandwidth: 3 nm; fluorescence bandwidth: 3 nm; response: 0.1 sec; sensitivity: middle; scanning speed: 200 nm / min

<MB3-C3-PE-Q>
The fluorescence intensity from 460 nm perylene in the excitation at 425 nm before and after adding MBt to MB3-C3-PE-Q is shown in the following table.

(Conditions for fluorescence measurement) Spectrofluorometer: FP-6500 manufactured by JASCO; excitation bandwidth: 3 nm; fluorescence bandwidth: 3 nm; response: 0.1 sec; sensitivity: middle; scanning speed: 200 nm / min

  As shown in Tables 11 to 13, by using anthraquinone for perylene, the quenching-luminescence phenomenon could be confirmed by supplying the target nucleic acid. Therefore, it was found that perylene can be used as a fluorophore and anthraquinone as a quencher. In particular, as shown in Table 13, stable quenching and strong luminescence could be confirmed by providing two perylenes and anthraquinones on the stem strands paired with the molecular beacons. From the above results, it can be seen that the quenching with the beacon closed contributes to the improvement of the signal intensity ratio. This is not because fluorescence quenching occurs only between perylene and anthraquinone forming a pair, but fluorescence quenching occurs due to the transfer of electrons through the natural base pair to the unpaired anthraquinone. It is considered a thing. That is, it was found that the two perylene-anthraquinone pairs introduced into the stem part do not function independently but synergistically contribute to stable quenching.

3. Confirmation of quenching and light emission by Cy3-MR <MB1-C3-Cy3-MR>
The fluorescence intensity from Cy3 at 563 nm in the excitation at 546 nm before and after adding MBt to MB1-C3-Cy3-MR is shown in the following table.

(Conditions for fluorescence measurement) Spectrofluorometer: FP-6500 manufactured by JASCO; excitation bandwidth: 3 nm; fluorescence bandwidth: 3 nm; response: 0.1 sec; sensitivity: middle; scanning speed: 200 nm / min

<MB2-C3-Cy3-MR>
The fluorescence intensity from Cy3 at 563 nm in the excitation at 546 nm before and after adding MBt to MB2-C3-Cy3-MR is shown in the following table.

(Conditions for fluorescence measurement) Spectrofluorometer: FP-6500 manufactured by JASCO; excitation bandwidth: 3 nm; fluorescence bandwidth: 3 nm; response: 0.1 sec; sensitivity: middle; scanning speed: 200 nm / min

  As shown in Tables 14 and 15, by using methyl red for Cy3, the quenching-luminescence phenomenon could be confirmed by supplying the target nucleic acid. Therefore, it was found that Cy3 can be used as a fluorophore and methyl red as a quencher.

(Synthesis of amidite monomers containing MRNO2)
Amidite monomers containing MRNO2 were synthesized according to the following scheme. 4-Amino-3-nitrobenzoic acid is dissolved in a mixed solvent of acetic acid and water, and an acidic solution of sodium nitrite in hydrochloric acid is added thereto, followed by stirring at 15-20 ° C. for 15 minutes. An aqueous solution was obtained. Next, while maintaining an acidic solution of hydrochloric acid of N, N-dimethylaniline at 0-5 ° C. on an ice bath, an aqueous solution of the above diazonium salt was dropped to obtain 4′-dimethylamino-2-nitroazobenzene-4-carboxylic acid. The acid (6-1) was obtained. Next, Nature Protocols, Vol. 2, pages 203 to 212 except that 4'-dimethylamino-2-nitroazobenzene-4-carboxylic acid (6-1) is used instead of azobenzene. The amidite monomer (6-2) was obtained according to the method described in 1.

Subsequently, molecular beacon-type oligonucleotide probes shown in Table 16 below were designed and synthesized. Various molecular beacons were synthesized according to the method described in Example 4 using the various amidide monomers and various fluorophores and quenchers synthesized or prepared in Examples 1-3. Various sequences are also shown.

  Abbreviations in Table 16 are as shown in the following figure. The abbreviations used in Tables 2 and 8 are also exactly the same as in the following figures.

For these beacons, changes in fluorescence intensity before and after the addition of the target nucleic acid MBt or MBt2 were observed. The conditions of the solution for measuring the fluorescence spectrum were as follows. The results are shown in Tables 17-20.
Concentration of beacon: 0.2 μmol / l
Concentration of target nucleic acid Nb: 0.4 μmol / l
Sodium chloride: 0.1 mol / l
Phosphate buffer: 10 mmol / l (pH 7.0)

<MB1-C3-Cy3-MRNO2>
The fluorescence intensity from 566 nm Cy3 in the excitation at 546 nm before and after adding MB-t to MB1-C3-Cy3-MRNO2 is shown in the following table.

(Conditions for fluorescence measurement) Spectrofluorometer: FP-6500 manufactured by JASCO; excitation bandwidth: 3 nm; fluorescence bandwidth: 3 nm; response: 0.1 sec; sensitivity: middle; scanning speed: 200 nm / min

The fluorescence intensity from 566 nm Cy3 in the excitation at 546 nm before and after adding MB-t2 to MB1-C3-Cy3-MRNO2 is shown in the following table.
(Conditions for fluorescence measurement) Spectrofluorometer: FP-6500 manufactured by JASCO; excitation bandwidth: 3 nm; fluorescence bandwidth: 3 nm; response: 0.1 sec; sensitivity: middle; scanning speed: 200 nm / min

<MB2-C3-Cy3-MRNO2>
The fluorescence intensity from 566 nm Cy3 in the excitation at 546 nm before and after adding MB-t to MB2-C3-Cy3-MRNO2 is shown in the following table.

(Conditions for fluorescence measurement) Spectrofluorometer: FP-6500 manufactured by JASCO; excitation bandwidth: 3 nm; fluorescence bandwidth: 3 nm; response: 0.1 sec; sensitivity: middle; scanning speed: 200 nm / min

The fluorescence intensity from 566 nm Cy3 in the excitation at 546 nm before and after adding MB-t2 to MB2-C3-Cy3-MRNO2 is shown in the following table.

(Fluorescence measurement conditions) Spectrofluorometer: JASCO FP-6500, excitation bandwidth: 3 nm; fluorescence bandwidth: 3 nm; response: 0.1 sec; sensitivity: middle; scanning speed: 200 nm / min

As shown in Tables 17 to 20, by using MRNO2 for Cy3, the quenching-luminescence phenomenon could be confirmed by supplying the target nucleic acid. Therefore, it was found that Cy3 can be used as a fluorophore and MRNO2 as a quencher. Moreover, the maximum absorption wavelength of Cy3 was 546 nm, MRNO2 was 513 nm, and the difference was 33 nm.
Furthermore, even if the target nucleic acid forms a double strand with the beacon fluorescent dye side strand as shown in Table 17 and Table 19, the beacon quencher side strand and double strand as shown in Table 18 and Table 20. Even when formed, the quenching-luminescence phenomenon could be confirmed.

In this example, the quenching ability associated with the association was evaluated for a model duplex capable of forming a fluorophore: quencher (1: 2) association. First, the following types of modified DNA were synthesized, and the degree of quenching when two quencher dyes were introduced in succession was observed.
Fluorescent chain 1a-Cy3 5'-GGTATC-Cy3-GCAATC-3 '
Quenching chain 1b-MR 3'-CCATAG-MR-CGTTAG-5 '
1b-NMR 3'-CCATAG-NMR-CGTTAG-5 '
1b-2 MR 3'-CCATAG-MR-MR-CGTTAG-5 '
1b-2NMR 3'-CCATAG-MR-MR-CGTTAG-5 '
1b-MR-NMR 3'-CCATAG-MR-NMR-CGTTAG-5 '
1b-NMR-MR 3'-CCATAG-NMR-MR-CGTTAG-5 '

  Next, buffer solution ([NaCl] = 100 mM, pH 7.0 10 mM phosphate buffer) so that the concentration of fluorescent chain 1a-Cy3 is 0.2 μM, and the quenching chain is twice the fluorescent chain (0.4 μM). And the fluorescence intensity from Cy3 at 564 nm was measured upon excitation at 546 nm. The results are shown in Table 21.

(Fluorescence measurement conditions) Spectrofluorometer: JASCO FP-6500; excitation bandwidth: 3 nm; fluorescence bandwidth: 5 nm; response: 0.1 sec; sensitivity: high; scanning speed: 200 nm / min; measurement Temperature: 20 ° C

  As shown in Table 21, it was found that the fluorescence of the fluorophore (Cy3) can be quenched more efficiently by introducing two identical or different quenchers successively into one fluorophore.

  In this example, the sensitivity of target nucleic acid detection by a double quencher type beacon was evaluated. Based on the result of Example 8, a beacon having the following configuration was designed and synthesized.

(beacon)
dqMB1-cy3-NMR 5'-TG- Cy3 -GTCCTTGAGAAAGGGCGAC- NMR - NMR -CA-3 '
dqMB2-cy3-NMR 5'-TGG- Cy3 -TCCTTGAGAAAGGGCGA- NMR - NMR -CCA-3 '
Mb1-cy3-NMR 5'-TG- Cy3 -GTCCTTGAGAAAGGGCGAC- NMR -CA-3 '
Mb2-cy3-NMR 5'-TGG- Cy3 -TCCTTGAGAAAGGGCGA- NMR -CCA-3 '
(Target nucleic acid)
Sba 3'-ACCAGGAACTCTTTCCCG-5 '
Sbb 3'-GAACTCTTTCCCGCTGGT-5 '

For these beacons, changes in fluorescence intensity were observed before and after adding the target nucleic acid Sba or Sbb. The conditions of the solution for measuring the fluorescence spectrum were as follows. Table 22 shows the results calculated from the fluorescence intensity at 564 nm upon excitation at 546 nm.
(Fluorescence spectrum measurement conditions) Concentration of beacon: 0.2 μmol / l; Concentration of target nucleic acid: 0.4 μmol / l; Sodium chloride: 0.1 mol / l; Phosphate buffer: 10 mmol / l (pH 7.0)

(Conditions for fluorescence measurement) Spectrofluorometer: FP-6500 manufactured by JASCO; excitation bandwidth: 3 nm; fluorescence bandwidth: 3 nm; response: 0.1 sec; sensitivity: middle; scanning speed: 200 nm / min; Measurement temperature: 20 ° C

  As described above, it showed a good S / N ratio regardless of the position of the fluorophore and quencher in the stem. In particular, it was found that Mb2-cy3-NMR, which had a low sensitivity, had a marked improvement effect on the S / N ratio by using a double quencher type (dbMB2-cy3-NMR).

Claims (13)

An oligonucleotide probe capable of forming a stem and a loop, comprising:
At least one fluorophore linked to a unit represented by the following formula (1) arranged between adjacent nucleotides of the stem;
(In the formula, X represents a fluorophore, 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. And Z represents a direct bond or a linking group.
At least two quenchers linked to a unit represented by the following formula (2) arranged at a site corresponding to the at least one fluorophore between adjacent nucleotides of the stem;
(In the formula, Y represents a quencher, 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.)
Comprising a probe.   The probe according to claim 1, wherein the fluorophore is arranged inside a nucleotide chain constituting a base sequence that specifically hybridizes with a target nucleic acid.   The fluorophore is any one selected from the group consisting of cyanine dyes, merocyanine dyes, condensed aromatic ring dyes, xanthene dyes, coumarin dyes and acridine dyes, and the quencher is selected from azo dyes. The probe according to claim 1 or 2, which is any one selected from the group consisting of:   The fluorophore is any one selected from Cy3, Cy5, thiazole orange, oxazole yellow, perylene, fluorescein, rhodamine, tetramethylrhodamine, Texas red, coumarin and acridine, and the quencher is methyl red, azobenzene And the probe according to claim 3, which is any one selected from methylthioazobenzene.   The probe according to claim 3, wherein the fluorophore is a condensed aromatic ring dye, and the quencher is anthraquinone or a derivative thereof.   6. The probe according to claim 5, wherein the fluorophore is perylene and the quencher is anthraquinone.   The probe according to claim 1, wherein one probe and one loop can be formed. A probe-immobilized body for detecting a target nucleic acid,
A solid support;
The oligonucleotide probe according to any one of claims 1 to 7, which has a base sequence capable of specifically hybridizing with at least a part of the target nucleic acid, and is held on the solid phase carrier;
An immobilization body.   The immobilized body according to claim 8, wherein the solid phase carrier is a plate-like body.   The immobilized body according to claim 8 or 9, comprising a plurality of oligonucleotide probes capable of respectively detecting a plurality of the target nucleic acids.   The immobilized body according to any one of claims 8 to 10, which is used for detection of SNP or gene mutation. A method for detecting a target nucleic acid, comprising:
The oligonucleotide probe according to any one of claims 1 to 7, wherein the oligonucleotide probe has a base sequence capable of specifically hybridizing with at least a part of the target nucleic acid, and A method of detecting a signal based on a fluorophore.   The method according to claim 12, wherein an immobilized body in which a plurality of the oligonucleotide probes capable of hybridization corresponding to a plurality of target nucleic acids are held on a solid phase carrier is used.