JP5071076B2 - Binding detection method using probe - Google Patents

Description

  The present invention relates to a probe that can bind to a predetermined substance. More specifically, the present invention relates to a probe to which an inclusion chemical species having optical activity is bound, and a binding detection method using the same.

  Currently, technologies for detecting genes (nucleic acids) and gene products (proteins) expressed in cells and tissues for the purpose of elucidating the molecular mechanisms of various diseases, establishing diagnostic methods, and searching for drug discovery targets. Have been developed.

  Among them, the detection method using a fluorescent dye generally obtains high sensitivity, and since it can be easily integrated in a small size such as a photodiode or a photomultiplier tube, and a detection device can be configured using inexpensive parts, etc. Currently most widely used.

  As an example, there is a method using an intercalating fluorescent dye that emits fluorescence when irradiated with excitation light in a state of being bound or inserted between base pairs of a double-stranded nucleic acid.

  In the presence of an intercalating fluorescent dye, a double strand formed when a hybridization (double strand formation) reaction between a nucleic acid strand to be detected and an oligonucleotide (probe) having a complementary base sequence is performed. An intercalating fluorescent dye is bound between the base pairs of the nucleic acid strand. At this time, the detection target nucleic acid chain can be detected by detecting the fluorescence emitted from the intercalating fluorescent dye by irradiation with excitation light.

  As another example, there is a method using a TaqMan probe (registered trademark, the same applies hereinafter) in which the 5 ′ end of an oligonucleotide is modified with a fluorescent dye and the 3 ′ end is modified with a quencher substance. This method is particularly used for real-time PCR.

  The TaqMan probe specifically hybridizes to the detection target DNA in the annealing step of the PCR reaction. At this time, since a quencher is present on the probe, the generation of fluorescence from the fluorescent dye is suppressed even when irradiated with excitation light. However, in the extension step, when the probe hybridized to the DNA to be detected is degraded by the 5 ′ → 3 ′ exonuclease activity of Taq DNA polymerase, the fluorescent dye is released from the probe, and the suppression by the quencher is released and the fluorescence is released. Is emitted. By detecting this fluorescence, it is possible to measure the amount of PCR amplification product produced and the amount of DNA to be detected in real time.

  Patent Document 1 discloses a hybridization detection method using an intercalating fluorescent dye. Patent Document 2 describes a method for quantitatively detecting human β-defensin 2 mRNA by a real-time PCR method using a TaqMan probe.

  Hereinafter, technical terms related to the present invention will be described.

  “Cyclodextrin” is a cyclic oligosaccharide obtained by allowing a certain enzyme to act on starch, and is a compound in which glucose is linked cyclically with α-1,4 bonds. Those in which 6, 7, and 8 glucoses are linked in a ring are called α-, β-, and γ-cyclodextrins, respectively. Also exist. At present, German Wacker Chemie has discovered an enzyme that selectively produces α-, β-, and γ-3 types of cyclodextrins, and is engaged in industrial production (see Non-Patent Document 1).

  The cyclodextrin has a bucket shape with no bottom, and the secondary hydroxyl group is located on the wide side of the ring and the primary hydroxyl group is located on the narrow side, so that the outside of the ring is hydrophilic. Inside is a place for hydrophobic cavities. Molecules, especially hydrophobic molecules in aqueous solution, are taken up into this cavity, ie "inclusion". The inclusion ability varies depending on the harmony between the size of the ring and the size of the molecule to be included and the hydrophobicity of the molecule.

  Since cyclodextrin is an oligosaccharide, it is water-soluble and can also be used to solubilize fat-soluble substances. Moreover, cyclodextrin is also optically active derived from the optically active oligosaccharide which is a structural unit.

  Inclusion phenomenon of cyclodextrin is an oral antifungal agent in the food, cosmetics and pharmaceutical industries, sustained release of volatile fragrances such as wasabi, stabilization of light and heat labile substances such as vitamins, etc. Sporanox (registered trademark) is used to improve water solubility. In the field of analytical chemistry, it is applied to affinity chromatography and capillary electrophoresis (see Non-Patent Document 2). Recently, application to drug delivery is considered promising (see Non-Patent Document 3).

  Research is also actively conducted on sensors for specific substances using the inclusion ability of cyclodextrins, that is, molecular recognition ability. Attempts have been made to detect guest compounds based on fluorescence intensity, fluorescence lifetime, etc. by adopting a polypeptide as a support for connecting functional parts such as fluorescent dyes, cyclodextrins, and quenchers as sensor probes (non-patented) Reference 4 and Non-Patent Document 5). Note that the above polypeptide is merely used as a support and cannot be bound to other substances. These techniques are for detecting the guest compound itself included in the cyclodextrin.

“Circular Dichroism (CD)” is a phenomenon in which a difference in absorbance occurs between right circularly polarized light and left circularly polarized light. The resulting polarized light is elliptically polarized light. The ellipticity is a function of the radiation intensity of right and left polarized light, and by substituting the Lambert-Beer equation, the relationship [θ] = 18000 / 4πlog ₁₀ e · Δε ≒ 3298Δε between the molar circular dichroism and the molar ellipticity Can be derived. That is, circular dichroism can be expressed by either molar circular dichroism or molar ellipticity. If the molar circular dichroism or molar ellipticity specific to a substance is known, the substance concentration can be calculated.

  Since cyclodextrin is optically active, it exhibits circular dichroism in the ultraviolet region according to the original absorption transition. When the originally non-optically active molecule G forms an inclusion or adduct with the optically active molecule H, it may exhibit circular dichroism depending on the absorption transition of G due to exciton coupling. This is called “induced circular dichroism”. If G is a pigment, an absorption transition appears in the visible part, and it is easy to detect it separately from other constituent species.

  Next, the “circular dichroism measuring device” will be described. A general circular dichroism measuring apparatus uses a non-polarized light mixed with light having various vibration vectors as excitation light, linearly polarized light by a polarizing prism, and then a circular polarization modulator such as a λ / 4 wavelength plate. To convert to left and right circularly polarized light. In synchronization with the left and right circularly polarized light, the absorbance data of the light that has passed through the sample is alternately collected by a detector such as a photomultiplier tube and processed. If the fluorescence of the sample is measured in synchronization with the left and right circularly polarized light, that is, the fluorescence detection circular dichroism is observed (see Patent Document 3).

JP 2006-296279 A JP 2004-248676 A Japanese Patent No. 3745881 "Old and New Food Material-Utilization Technology of Natural Cyclodextrins", Foods Food Ingredients Journal. Japan, 2005, Vol.210, No.3, p.222-243 “Cyclodextrinsand Their Applications in Analytical Chemistry.”, Chemical Reviews, 1992, Vol.92, No.6, p.1457-1470 "Nanomaterial cyclodextrin", edited by Cyclodextrin Society, Sangyo Tosho, 2005 “Guest-Induced Diminishment in Fluorescence Quenching and Molecule Sensing Ability of A novel Cyclodextrin-Peptide Conjugate.” Journal of American Chemistry Society, 2001, Vol.123, No.30, p.7435-7436 “Fluorescent Cyclodextrin / Peptide Hybrids with a Novel Guest-Responsive Chemosensor in the Peptide Side Chain.” Macromolecular Raped Communication, 2002, Vol.23, No.15, p.905-908

  The above-described method using an intercalator fluorescent dye can use an inexpensive intercalator fluorescent dye, and is excellent in cost. However, there has been a problem that detection sensitivity decreases due to background fluorescence from a free dye not bound to a double strand and noise fluorescence from a dye bound to a single strand having a hairpin structure or a mismatched strand. In particular, when detecting a very small amount of nucleic acid chain, it has been difficult to obtain sufficient sensitivity.

  The method using the TaqMan probe can solve the problems of background fluorescence and noise fluorescence that have occurred with intercalating fluorescent dyes, and can perform detection with higher sensitivity. However, since the TaqMan probe is very expensive, there is a problem in cost. In addition, since the fluorescent dye is directly labeled on the probe in the method using the TaqMan probe, compared to the method using an intercalator fluorescent dye that detects fluorescence by introducing an excessive amount of fluorescent dye into the reaction system. There is also a problem that it is easily affected by the deterioration of the fluorescent dye during fluorescence detection.

  Therefore, a main object of the present invention is to provide a novel probe that is excellent in cost and can obtain high detection sensitivity, and a binding detection method using the same.

In order to solve the above-described problems, the present invention provides a molecular species that can include other chemical species in a molecular chain that can bind to a predetermined substance, and the first chemical species that has optical activity is bound thereto. A probe is provided.
In the probe according to the present invention, a second chemical species that can be included in the first chemical species may be further bonded to the molecular chain.
In this case, the first chemical species and the second chemical species are configured to maintain an inclusion state when not bonded to the substance, and to be released from the inclusion state upon binding with the substance. . This inclusion state can be configured to be stabilized by, for example, a stem loop structure formed by molecular chains.
The binding reaction between the probe and the substance is performed in the presence of a dye that can be included in the first chemical species, and the first chemical species and the second chemical species that are maintained when the probe and the substance are not bound to each other. By detecting the induced circular dichroism that is acquired by inclusion of the dye with the first chemical species by releasing the inclusion state of the probe by the binding of the probe and the substance, the binding of the substance to the probe is detected. Can be detected.
At this time, it is desirable that the inclusion ability of the dye with respect to the first chemical species is lower than the inclusion ability of the second chemical species with respect to the first chemical species.

The second chemical species of the probe according to the present invention may be a dye.
By performing the binding reaction between the probe and the substance, the inclusion state of the first chemical species and the dye maintained when the probe and the substance are not bonded is released by the binding of the probe and the substance, thereby the dye. By detecting that the induced circular dichroism disappears, the binding between the substance and the probe can be detected.

Furthermore, the present invention provides the above first compound wherein a chemical species capable of including another chemical species and a first chemical species having optical activity are bound to a molecular chain capable of binding to a predetermined substance. In addition to the above-mentioned probe, a probe pair comprising a second probe in which a second chemical species that can be included in the first chemical species is bound to a molecular chain is provided.
In this probe pair, the first chemical species and the second chemical species are configured to maintain the inclusion state when not bonded to the substance and to be released from the inclusion state when bonded to the substance. The
The second chemical species may be a dye, which performs a binding reaction between the probe pair and the substance, and the inclusion state of the first chemical species and the dye maintained when the probe pair and the substance are not bonded is By detecting the disappearance of the induced circular dichroism of the dye by being released by the binding between the probe and the substance, the binding between the substance and the probe can be detected.
The binding detection method according to the present invention can be suitably used particularly for the real-time PCR method.

  Here, in the present invention, the “predetermined substance” includes at least nucleic acids, peptides, and proteins. Accordingly, the combination of “predetermined substance” and “molecular chain capable of binding to the predetermined substance” includes, for example, a specific single-stranded nucleic acid and a nucleic acid chain or a nucleic acid-like structure chain that hybridizes thereto, a specific protein, and the like. There are combinations of proteins or peptides that bind to this. In addition, it is possible to combine specific nucleic acids and peptides, compounds that interact with proteins, specific substances and substances that interact with this due to pi-electron interactions, hydrogen bonds, electrostatic effects, etc. is there.

  “Induced circular dichroism” refers to a phenomenon in which when a chiral substance absorbs circularly polarized light, a difference in absorbance occurs between left circularly polarized light and right circularly polarized light. Induced circular dichroism occurs only at wavelengths that the material absorbs. Induced circular dichroism is achieved by converting non-polarized excitation light into left and right circularly polarized light using a circular polarization modulator such as a polarizing prism and a λ / 4 wavelength plate, and irradiating the material. It can detect by detecting the light-absorbency difference with respect to.

  In addition, the “stem loop structure” generally means that, in single-stranded DNA or single-stranded RNA, when there are complementary sequences in two regions separated in the molecule, A double strand is formed by interaction, and a random sequence sandwiched between the two regions forms a loop structure. Also called hairpin loop. The three-dimensional (three-dimensional) structure of a protein or peptide is determined by the presence of a hydrophilic part and a hydrophobic part in the molecule, and by interactions between hydrogen bonds and pi electrons between molecules. In the “stem loop structure” of the present invention, in addition to the hairpin loop in the nucleic acid chain described above, in the three-dimensional structure of proteins and peptides, as a result of interaction between two distant regions in the molecule, It is assumed that the sandwiched arrangement part has a loop-like structure. In addition to this, a predetermined molecular chain is a hydrogen bond between two regions in the molecule, an electrostatic effect, a pi electron, etc. It is possible to widely include a loop-like structure formed as a result of the interaction by.

  INDUSTRIAL APPLICABILITY According to the present invention, a novel probe that is excellent in cost and can obtain high detection sensitivity, and a binding detection method using the same are provided.

  DESCRIPTION OF EXEMPLARY EMBODIMENTS Hereinafter, preferred embodiments for carrying out the invention will be described with reference to the drawings. In addition, embodiment described below shows an example of typical embodiment of this invention, and, thereby, the range of this invention is not interpreted narrowly.

  FIG. 1 is a schematic diagram showing a first embodiment of a probe according to the present invention. In the figure, the probe is denoted by reference numeral 1.

  In FIG. 1, symbol P indicates a molecular chain that can bind to a predetermined substance. The molecular chain P is a binding region that recognizes and binds to a specific structure of a substance by self-assembly, adsorption, light irradiation, heating, or the like. The molecular chain P can be constituted by a polymer chain having a chemically active group such as an oligonucleotide, an oligopeptide, or a polypeptide depending on the substance.

  In FIG. 1, symbol H denotes a first chemical species (hereinafter referred to as “host H”) that is a chemical species that can include other chemical species (inclusion chemical species) and has optical activity. . It is desirable to use a host H having a small overlap between its light absorption spectrum (hereinafter simply referred to as “absorption spectrum”) and an absorption spectrum of dye D described later. This is for detecting the induced circular dichroism of the dye D (this point will be described later).

Host H, in Figure 1, the spacer denoted by reference numeral S _1, is coupled to one end of the molecular chain P. Spacer S ₁ may typically be a bifunctional spacer for use in the chemical modification, such as oligonucleotide probes, e.g., alkylene or oligopeptides, oligonucleotides, polyether, composed of a polymer structure such as polyvinylene. The spacer S ₁ may include an ether bond, a sulfide bond, a peptide bond, an ester bond, a urethane bond, a phosphate bond, a sulfonic acid, an arylene, or the like, or may have a branched structure. Furthermore, arylene may contain heteroatoms.

  In FIG. 1, symbol A is a second chemical species that can be included in the host (hereinafter referred to as “anchor A”). The anchor A is included in the host H and inhibits the later-described dye D from being included in the host H. In order to ensure the effect of the anchor A on the inclusion of the dye D, the inclusion ability of the anchor A with respect to the host H is desirably equal to or higher than that of the dye D with respect to the host H.

Anchor A is, in FIG. 1, the spacer denoted by reference numeral S _2, is coupled to the host H and the opposite end of the molecular chain P. Structure of the spacer S ₂ is may be similar to the spacer S _1, not necessarily identical, can be selected from among those described in the spacer S ₁ arbitrarily.

  FIG. 2 is a diagram illustrating an example of chemical species that can be suitably employed as the host H.

In FIG. 2, (A) is an α-cyclodextrin substituent, (B) is a β-cyclodextrin substituent, (C) is a γ-cyclodextrin substituent, and (D) is heptakis (o-methyl) -γ-. Cyclodextrin substituent is shown. In the figure, "-X-" is "-O-", "-S-", "-NH-", "-OC (= O)-", "-OS (= O) _2- ", "OP (= O) ₂ O— ”and“ —NHC (═O) — ”.

  In each chemical species, all or some of the hydroxyl groups are converted into mercapto groups, oxy groups, thio groups, amino groups, carbamoyl groups, oxycarbonyl groups, carboxy groups, sulfo groups, phosphate groups, trialkylsiloxy groups, etc. It may be.

  FIG. 3 is a diagram showing an example of chemical species that can be suitably employed as the anchor A.

In FIG. 3, (A) is a cholesterol substituent, (B) is a lithocol substituent, (C) is an adamantane substituent, (D) is a vitamin A substituent, (E) is a ubiquinone substituent, and (F) is naphthalene. Substituents, (G) represents a pyrene substituent, (H) represents a benzo [c] phenanthrene substituent, (I) represents a binaphthyl substituent, and (J) represents a ruthenium (II) tris (phenanthroline) substituent. In the figure, "-X _2- " means "-O-", "-S-", "-NH-", "-OC (= O)-", "-OS (= O) _2- ", It is a group selected from “OP (═O) ₂ O—” and “—NHC (═O) —”.

  Each chemical species may have a chiral structure in the molecule. In addition, chemical species having a steroid structure can be widely adopted as the anchor A.

  FIG. 4 is a schematic diagram illustrating a specific configuration example of the probe 1.

  In FIG. 4, when the predetermined substance is a nucleic acid, the probe denoted by reference numeral 11 is a molecular chain P as an oligonucleotide (base number n) having a base sequence complementary to the nucleic acid, and a host H as β- The case where cyclodextrin and anchor A are lithocholic acid is shown.

  FIG. 5 is a schematic diagram showing the behavior of the probe 1. (A) shows a state at the time of non-bonding with a predetermined substance, and (B) shows a state at the time of binding.

  First, in the probe 1 at the time of non-bonding with a predetermined substance shown in FIG. 5A (see symbol T in FIG. 5B), the molecular chain P is bent by the inclusion of the anchor A by the host H. The loop structure shown in the figure is taken.

In the probe 1, in the molecular chain P, two regions (“stem portions St ₁ and St ₂ ” described later) located at both ends of the spacer S ₁ side and the spacer S ₂ side are hydrogen-bonded (FIG. 5A). ), And the formation of the loop structure of the molecular chain P is promoted and stabilized. That is, when the anchor A is included in the host H and the molecular chain P is bent, the two regions located at both ends of the spacer S ₁ side and the spacer S ₂ side form hydrogen bonds B to each other, thereby It is constructed so that the stem loop structure of chain P is stabilized.

  On the other hand, in the probe 1 at the time of binding with the substance T shown in FIG. 5 (B), the molecular chain P cannot take a loop structure due to the binding with the substance T. Therefore, the anchor A cannot easily approach the host H to a distance at which an inclusion state can be formed, and the probability of forming an inclusion state with the host H is extremely reduced.

  As described above, the probe 1 is characterized in that the host H and the anchor A maintain the inclusion state at the time of non-bonding with the substance T, and the inclusion state is released by the binding with the substance T.

FIG. 6 is a schematic diagram showing a stem loop structure formed by the molecular chain P. (A) shows the spacers S ₁ and S ₂ and the molecular chain P when the stem-loop structure is not formed, and (B) shows the enlarged spacers S ₁ and S ₂ and the molecular chain P when formed. In the figure, the probe 11 having the molecular chain P as an oligonucleotide is illustrated.

The molecular chain P shown in FIG. 6A is provided with stem portions St ₁ and St _{2 in} two regions located at both ends of the spacer S ₁ side and the spacer S ₂ side. The base sequence of the stem part St ₁ is “GATCGAA” from the 5 ′ end side after the spacer S ₁ , and the base sequence of the stem part St ₂ is “TTCGATC” from the 5 ′ end side toward the spacer S _2. ing.

As described above, when the anchor A is included in the host H, the molecular chain P is bent to form a loop structure. At this time, as shown in FIG. 6 (B), the stem portion St ₂ is disposed in the opposite direction to the stem portion St _1. The base sequence “TTCGATTC” of the stem portion St ₂ is made complementary to the base sequence “GATCGAA” of the stem portion St ₁ in this arrangement state. Thus, it is possible to form hydrogen bonds B between the base opposite the stem portion St ₁ and St _2.

  Due to the formation of this hydrogen bond B, the molecular chain P forms a stem loop structure, and the formation of the loop structure of the molecular chain P by inclusion of the anchor A with the host H is further promoted and stabilized. Become.

This stem loop structure promotes and stabilizes the formation of the loop structure of the molecular chain P. The strength and number of hydrogen bonds between the stem parts St ₁ and St ₂ can be arbitrarily determined according to the molecular species and number of molecules used in the molecular chain P. By setting to, it can be arbitrarily adjusted.

When the molecular chain P is configured as an oligonucleotide, the number of base sequences in the loop part (the region between the stem part St ₁ and the stem part St _{2 in} the molecular chain P) is preferably about 4 to 8 bases. Yes ("A Semiflexible Polymer Model Applied to Loop Formation in DNA Hairpins." Biophysical Journal 2001, Vol.81, p2864-2875
reference)

  This stem-loop structure is intended to ensure that the inclusion state of the host H and the anchor A is maintained at the time of non-bonding with the substance T, and is essential for inclusion of the anchor A to the host H. It is not the composition of. As described above, the loop structure of the molecular chain P occurs as a result of the inclusion of the anchor A with the host H, and the hydrogen bond B functions to promote and stabilize the formation of this stem loop structure.

  FIG. 7 is a schematic diagram illustrating a method for detecting the binding between the probe 1 and the substance T. (A) shows the reaction system before the binding reaction between the probe 1 and the substance T, and (B) shows the reaction system after the binding reaction.

  First, as shown in FIG. 7A, the binding reaction between the substance T and the probe 1 is performed in the presence of a dye that can be included in the host H (see symbol D in the figure).

  In FIG. 7, the symbol N represents another substance that coexists with the substance T. Since the molecular chain P of the probe 1 does not bind to the other substance N, the probe 1 and the other substance N do not bind. In the figure, the probe 1, the substance T, the other substance N, and the dye D are shown one molecule at a time for convenience, but a plurality of these exist in the reaction system.

  As shown in the figure, the host H maintains the inclusion state with the anchor A before the binding reaction. For this reason, although the dye D has an inclusion ability with respect to the host H, since the anchor A inhibits the inclusion of the dye D with the host H, the probability that the dye D is included with the host H can be ignored. Hereinafter, the dye D that is not included in the host H is referred to as “free dye D”.

When the non-polarized light is converted into linearly polarized light by a polarizing prism and then converted to circularly polarized light by a λ / 4 wavelength plate (see symbol L _{1 in the} figure), free dye D is irradiated by free dye D. (in the figure, reference numeral L ₂₎ specific wavelength absorbed transmitted light measurement object radiation occurs as.

At this time, since the free dye D does not have optical activity (hereinafter also referred to as “chirality”), there is no difference between the absorbance of the left circularly polarized light and the right circularly polarized light absorbed by the dye D. Therefore, the induced circular dichroism does not occur when the measurement light L ₁ is irradiated to the dye D, and the induced circular dichroism is not detected by the detection of the measurement target light L ₂ .

  In contrast, after the binding reaction shown in FIG. 7B, the inclusion state between the host H and the anchor A is released by the binding between the molecular chain P and the substance T, and the dye D is substituted for the anchor A. Is included in host H. The dye D is induced by a chiral field provided by the host H by inclusion in the host H and acquires chirality.

Therefore, as shown in the figure, when the measurement light L ₁ is irradiated to the dye D included in the host H, the absorbance difference between the left circularly polarized light and the right circularly polarized light is caused by the chirality at the absorption wavelength of the dye D, the fact that the target light L _3, which exhibits a circular dichroism is detected.

Thus, in the probe 1, the transmitted light (measurement target light L ₂ ) does not exhibit induced circular dichroism when not coupled to the substance T. However, when the probe 1 is coupled to the substance T, the transmitted light (measurement target light) L ₃ ) exhibits induced circular dichroism. Therefore, the binding reaction between the substance T and the probe 1 is performed in the presence of the dye D, and the induced circle of the transmitted light (the measurement target light L ₂ or the measurement target light L ₃ ) generated from the dye D by the irradiation of the measurement light L ₁ By detecting dichroism, it becomes possible to detect the binding between the substance T and the probe 1.

The dye D is not particularly limited as long as it is a dye that can be included in the host H. More preferably, the inclusion ability of the dye D with respect to the host H is less than or equal to the inclusion ability of the anchor A with respect to the host H. Things are desirable. Thereby, before the binding reaction (see FIG. 7A), the anchor A can surely inhibit the inclusion of the dye D to the host H. In addition, for the detection of the measurement target light L ₂ and the measurement target light L ₃ , it is desirable that the dye D has a small overlap between the absorption spectrum and the absorption spectrum of the host H. When the molecular chain P of the probe 1 is an oligonucleotide, it is preferable to use a dye having no intercalator property.

  FIG. 8 is a diagram showing an example of chemical species that can be suitably employed as the dye D.

  In FIG. 8, (A) is a trimethine cyanine dye (Cy3 skeleton), (B) is a pentamethine cyanine dye (Cy5 skeleton), (C) is thiazole orange (TO), (D) is acridine orange (AO), (E) is acridine red (AR), (F) is neutral red (NR), (G) is dansylic acid (DA), (H) is rhodamine B (RhB), (I) is fluorescein (FL), ( J) represents ethyl bromide (EB), (K) represents pyrene, (L) represents coumarin 102 (C102), and (M) represents ethyl red (ER). Moreover, these pigment | dyes may have a substituent.

Detection of induced circular dichroism in the measurement target light L ₂ and the measurement target light L ₃ can be performed by the general circular dichroism measurement apparatus described above. Specifically, non-polarized light mixed with various vibration vectors is used as excitation light, converted into linearly polarized light by a polarizing prism, and then left and right circularly polarized light by a circular polarization modulator such as a λ / 4 wavelength plate. Convert to In synchronization with this left and right circularly polarized light, the absorbance data of the transmitted light that has passed through the sample is alternately collected by a detector such as a photomultiplier tube, and the difference in absorbance between the left and right polarized light is calculated to detect the induced circular dichroism. I do.

  In this case, if the pickup for magneto-optical recording is modified in order to reduce the size and increase the sensitivity of the measuring device, it can be used as a detector. Reading (ie, detection) in magneto-optical recording is essentially a phenomenon in which linearly polarized light is incident on a magnetized medium, and the polarization plane of the reflected light is slightly rotated with respect to the incident light to become elliptically polarized (magnetic) This is called the Kerr effect. Here, if a sample showing circular dichroism is arranged instead of the magnetic medium, the ellipticity with respect to incident light can be measured in the same manner.

  A pickup for magneto-optical recording generally includes a semiconductor laser, a plurality of photodetectors, a polarization beam splitter or a polarization hologram element, and an objective lens. The light emitted from the semiconductor laser is condensed on the medium by the objective lens, and the reflected light or transmitted light is separated into right and left polarized light by the polarization beam splitter or the polarization hologram element, and reaches each photodetector. The ellipticity can be calculated from the intensity of right and left polarized light.

Further, so far, the case where the induced circular dichroism of the transmitted light (measurement target light L ₂ or measurement target light L ₃ ) exhibited by the dye D by irradiation of the measurement light L ₁ has been described, but as an alternative method The fluorescence amount of the dye D excited by the left and right circularly polarized light separated by the circularly polarized light modulator is collected in synchronism with the circularly polarized light modulator and processed, and induced circular dichroism ("fluorescent circle 2" It is also possible to detect “color”.

  In this case, since there is a difference in the left and right circularly polarized light absorbed by the dye D placed in the chiral field, a difference also occurs in the amount of fluorescence excited by each. On the other hand, since the free dye D absorbs right and left circularly polarized light equally, the amount of fluorescence is the same. Therefore, if this difference is detected, the dye D placed in the chiral field can be distinguished from the free dye D.

  Next, a method for detecting the binding between the probe 1 and the substance T will be described more specifically based on FIG.

  Here, a case where a nucleic acid (hereinafter referred to as “detection target nucleic acid T”) is detected as the substance T using the above-described probe 11 in which the molecular chain P is configured as an oligonucleotide (base number n) will be described as an example.

  First, as shown in FIG. 7A, the hybridization reaction between the sample containing the detection target nucleic acid chain T and the probe 11 is performed in the presence of the dye D. For example, this hybridization reaction may be performed in PCR or a DNA microarray (DNA chip). In PCR, the probe 11 is used in place of the conventional Taqman probe, and a hybridization reaction between the amplified nucleic acid chain T to be detected and the probe 11 is performed during the annealing step. In the case of a DNA microarray, the probe 11 is solid-phased on a substrate such as a slide glass, and a hybridization reaction with the nucleic acid chain T to be detected contained in the sample is performed. The immobilization of the probe 11 will be described later with reference to FIG.

In these hybridization reactions, at the stage where the probe 11 and the nucleic acid strand T to be detected do not form a double strand, since the host H maintains the inclusion state with the anchor A, the dye D is included in the host H. First, the probability that the induced circular dichroism is detected by detecting the measurement target light L ₂ is extremely small (see FIG. 7A).

In contrast, when the probe 11 and the nucleic acid strand T to be detected form a double strand, the inclusion state between the host H and the anchor A is released, and the dye D is included in the host H. Accordingly, the dye D acquires chirality, and the induced circular dichroism is detected by detecting the measurement target light L ₃ (see FIG. 7B).

Therefore, according to the probe 11, by detecting the induced circular dichroism measurement target light L ₃ at the annealing step of PCR, detecting the hybridization between the detection target nucleic acid strand T and probe 11 of the PCR solution Can do. Furthermore, based on the difference in absorbance of the light to be measured L ₃ (with induced circular dichroism) from the left and right circularly polarized light exhibited by the dye D included in the host H, the amount of amplification of the nucleic acid chain T to be detected is quantitatively detected in real time. can do.

  Formation of the inclusion state of host H and dye D in PCR can also be carried out by decomposing the probe using the 5 ′ → 3 ′ exonuclease activity of polymerase as is done with conventional Taqman probes. It is. Specifically, the probe 11 hybridized to the nucleic acid strand T to be detected in the annealing step is decomposed by the exonuclease activity of the polymerase in the extension (extension reaction) step, and the host H is released from the probe 11 body. Thus, after the extension step, the liberated host H and the free dye D contained in the reaction solution form an inclusion state, and the induced circular dichroism is detected based on the difference in absorbance of the dye D with respect to the left and right circularly polarized light. it can.

Also in DNA microarrays, on the basis of the induced circular dichroism in the measurement target light L _3, it is possible to detect the hybridization between the probe 11 immobilized on the substrate and detected nucleic acid strand T. Then, the measurement target light L ₂ generated from the free dye D (without induced circular dichroism) and the measurement target light L ₃ generated from the dye D included in the host H (with induced circular dichroism, elliptically polarized light) ) And the target nucleic acid chain T in the sample can be quantitatively detected.

As described above, according to the probe 1, based on the induced circular dichroism measurement target light L _3, since it is possible to detect the binding of substance T and the probe 1, the conventional intercalator Background fluorescence and noise fluorescence that are generated by the detection method using a fluorescent dye are not generated, and high detection sensitivity can be obtained.

  In addition, the use of an inexpensive dye D can solve the cost problem of the TaqMan probe. Furthermore, by introducing a large excess amount of inexpensive dye D into the reaction system, it is possible to solve the problem of fluorescent dye degradation during fluorescence detection, which has been caused by the method using the TaqMan probe.

  Next, another specific example of the method for detecting the binding between the probe 1 and the substance T will be described with reference to FIGS.

  In FIG. 9, the probe denoted by reference numeral 12 shows a case where the molecular chain P is a peptide (number of amino acids 14) that can bind to the protein when the substance T is a protein.

  In the lower part of FIG. 9, the peptide sequence of the molecular chain P is shown enlarged. The molecular chain P is a peptide sequence represented by “Leu-Lys-Lys-Leu-Leu-Lys-Leu-Leu-Lys-Lys-Leu-Leu-Lys-Leu” and forms an α-helix structure. It has a sequence to do. Hereinafter, a method for detecting the binding of calmodulin (hereinafter referred to as “calmodulin T”), which is a protein that recognizes and binds to the α-helix structure, to the probe 12 using the probe 12 will be described.

  First, the binding reaction between calmodulin T and the probe 12 is performed in the presence of the dye D. Such a binding reaction between calmodulin T and the probe 12 can be viewed as an interaction between proteins and can be used for functional analysis of the protein.

As shown in FIG. 10A, at the stage where the probe 12 and calmodulin T are not bound, the presence of host H and anchor A in the same molecule makes it possible to gain free energy, particularly entropy, due to intramolecular complex formation. Because the host H maintains the inclusion state with the anchor A due to the gain of the dye D, the dye D is difficult to be included in the host H, and the probability that the induced circular dichroism is detected by detecting the measurement target light L ₂ is extremely low. .

On the other hand, as shown in FIG. 10B, when calmodulin T recognizes the α-helix structure formed by the molecular chain P of the probe 12 and binds to the probe 12, the host H and the anchor A The inclusion state is released, and the dye D is included in the host H. As a result, the dye D acquires chirality, and the induced circular dichroism is detected by detecting the measurement target light L ₃ .

Therefore, according to the probe 12, by detecting the induced circular dichroism measurement target light L ₃ in the coupling reaction, it is possible to detect the binding between the probe 12 and the calmodulin T. Further, based on the difference in absorbance of the light to be measured L ₃ (with induced circular dichroism) exhibited by the dye D included in the host H with respect to left and right circularly polarized light, the amount of the probe 12 bound to calmodulin T is quantitatively determined. Can be detected.

  This method for detecting binding between calmodulin T and probe 12 can be applied to detection of binding between various proteins and probe 12 in place of calmodulin by arbitrarily designing the peptide sequence of molecular chain P. This allows protein functional analysis such as screening peptide sequences (antigens) that can be recognized by the target protein from the synthetic peptide library, and screening proteins that can bind to the specified peptide sequence from the protein library. Can be done.

  FIG. 11 is a schematic diagram showing a second embodiment of the probe according to the present invention. In the figure, the probe is denoted by reference numeral 2.

In FIG. 11, reference symbol P denotes a molecular chain that can bind to a predetermined substance, reference symbol H denotes a chemical species that can include other chemical species (inclusion chemical species), and the first chemical compound that has optical activity. Species (host), symbols S ₁ and S ₂ represent spacers. The molecular chain P, host H, spacers S ₁ and S ₂ can have the same configuration as that of the probe 1 shown in FIG.

  Here, in probe 1, anchor A is bound to one end of molecular chain P as a second chemical species that can be included in host H (see FIG. 1), whereas in probe 2 shown in the figure. The second chemical species is different in that dye D is bound. This dye D is the same as that described in FIG. 7, and is a dye that can be included in the host H (see FIG. 8).

The probe 2 is configured such that the spacer S ₃ is bonded to the opposite side of the spacer S ₁ of the molecular chain P so that the probe 2 can be bonded to another substance. For example, when the probe 2 is used for the DNA microarray mentioned above, the spacer S ₃ is necessary for immobilizing the probe 2 on the substrate surface.

The spacer S ₃ may typically be a bifunctional spacer which is used for immobilizing the substrate surface oligonucleotide probes and the like. As the substrate, one obtained by molding silicon or glass, or one obtained by surface-treating these with gold, polymer, or diamond-like carbon is used. Alternatively, the polymer film itself can be used as the substrate. As the spacer S ₃ , a spacer having an appropriate active group is used according to the binding target substance on the substrate surface.

Table 1 exemplifies combinations of the active group at the end of the spacer S _{3 and} the active group of the binding target substance on the substrate surface, and the binding method at that time.

  FIG. 12 is a schematic diagram illustrating a specific configuration example of the probe 2.

  In the figure, when the predetermined substance is a nucleic acid, the probe denoted by reference numeral 21 has a molecular chain P as an oligonucleotide (base number n) having a base sequence complementary to the nucleic acid, and a host H as β- The case where cyclodextrin and dye D are rhodamine B is shown.

  FIG. 13 is a schematic diagram showing the behavior of the probe 2. (A) shows a state at the time of non-bonding with a predetermined substance, and (B) shows a state at the time of binding.

  In the probe 2 at the time of non-bonding with the predetermined substance shown in FIG. 13 (A) (see symbol T in FIG. 13 (B)), the molecular chain P is bent by inclusion of the dye D in the host H. The loop structure is as follows.

Also in the probe 2, like the probe 1, the spacers S ₁ and S ₂ form a hydrogen bond (see symbol B in FIG. 13A), thereby promoting and stabilizing the formation of the loop structure of the molecular chain P. It is configured to be

  In the probe 2 at the time of binding to the substance T shown in FIG. 13B, the molecular chain P cannot take a loop structure due to the binding to the substance T. Therefore, the dye D cannot approach the host H to a distance that can form an inclusion state, and does not form an inclusion state with the host H.

  As described above, the probe 2 is characterized in that the host H and the dye D maintain the inclusion state at the time of non-bonding with the substance T, and the inclusion state is released by the binding with the substance T.

  FIG. 14 is a schematic diagram illustrating a method for detecting the binding between the probe 2 and the substance T. (A) shows the reaction system before the binding reaction between the probe 1 and the substance T, and (B) shows the reaction system after the binding reaction.

  First, the binding reaction between the substance T and the probe 2 is performed. Here, unlike the binding detection method using the probe 1 described in FIG. 7, in the probe 2, since the dye D is bound to the probe itself, it is not necessary to newly add the dye D to the reaction system.

  In FIG. 14, the symbol N represents another substance that coexists with the substance T. Since the molecular chain P of the probe 2 cannot bind to another substance N, the other substance N and the probe 1 do not bind. In the figure, the probe 2, the substance T, and the other substance N are shown one molecule at a time for convenience, but a plurality of these exist in the reaction system. Moreover, in the figure, the symbol K represents the substrate surface on which the probe 2 is solid-phased (this will be described later).

  As shown in FIG. 14A, the host H maintains the inclusion state with the dye D before the binding reaction with the substance T. In this state, the dye D is induced by the chirality of the host H and has chirality.

Therefore, as shown in the figure, when the measurement light L ₁ is irradiated to the dye D included in the host H, the absorbance difference between the left circularly polarized light and the right circularly polarized light is caused by the chirality at the absorption wavelength of the dye D, Measurement target light L ₃ exhibiting circular dichroism is detected. The measurement light L ₁ is as described in FIG.

  On the other hand, after the binding reaction with the substance T shown in FIG. 14B, the inclusion state between the host H and the dye D is released by the binding between the molecular chain P and the substance T. Due to the release of the inclusion state, the dye D loses the chirality induced by the host H.

Therefore, even if the measurement light L ₁ is irradiated to the dye D, there is no difference between the absorbance of the left circularly polarized light and the right circularly polarized light absorbed by the dye D, and the induced circular dichroism is detected by detecting the measurement target light L _2. It will not be detected.

As described above, in the probe 2, the reflected light (measurement target light L ₃ ) exhibits induced circular dichroism when not coupled to the substance T, whereas the reflected light (measurement target light) is coupled to the substance T. induced circular dichroism of L ₂₎ is lost. Therefore, the binding reaction between the substance T and the probe 2 is performed, and the induced circular dichroism of the reflected light (measurement target light L ₂ and measurement target light L ₃ ) generated from the dye D upon irradiation with the measurement light L ₁ is detected. As a result, the binding between the substance T and the probe 2 can be detected.

  Next, a method for detecting the binding between the probe 2 and the substance T will be described more specifically based on FIG.

  Here, a case where a nucleic acid (hereinafter referred to as “detection target nucleic acid T”) is detected as the substance T using the above-described probe 21 (see FIG. 12) in which the molecular chain P is configured as an oligonucleotide (base number n) is used. This will be described as an example.

  First, a hybridization reaction between the sample containing the nucleic acid chain T to be detected and the probe 21 is performed. In the figure, the hybridization reaction in the DNA microarray in which the probe 21 is immobilized on the substrate surface indicated by the symbol K is illustrated.

  In the hybridization reaction, at the stage where the probe 21 and the nucleic acid strand T to be detected do not form a double strand, the host H maintains the inclusion state with the dye D. Color is detected (see FIG. 14A).

In contrast, when the probe 21 and the detection target nucleic acid strand T forms a duplex, it is released inclusion state of the host H and the dye D, dye D is to lose chirality of the target light L ₂ The induced circular dichroism is not detected by the detection (see FIG. 14B).

Therefore, according to the probe 21, on the basis of the induced circular dichroism in the measurement target light L _3, it is possible to detect the hybridized probe 21 immobilized on the substrate surface K and the detection target nucleic acid strand T . Further, the detection target nucleic acid chain T in the sample is quantitatively detected based on the difference in absorbance with respect to the left and right circularly polarized light of the measurement target light L ₃ (with induced circular dichroism) exhibited by the dye D included in the host H. It becomes possible.

Moreover, if PCR is performed using the probe 21 instead of the conventional Taqman probe and the induced circular dichroism of the measurement target light L ₃ is detected in the annealing step, the amplified detection target nucleic acid chain T and the probe 21 are detected. Hybridization can be detected. Then, based on the difference in absorbance of the measurement target light L ₃ (with induced circular dichroism) exhibited by the dye D included in the host H with respect to the left and right circularly polarized light, the amount of amplification of the detection target nucleic acid chain T is quantitatively detected in real time. can do. Furthermore, as described above, the inclusion state between the host H and the dye D can be released by decomposing the probe using the 5 ′ → 3 ′ exonuclease activity of the polymerase.

As described above, according to the probe 2, based on the induced circular dichroism measurement target light L _3, since it is possible to detect the binding of substance T and the probe 2, the conventional intercalator Background fluorescence and noise fluorescence that are generated by the detection method using a fluorescent dye are not generated, and high detection sensitivity can be obtained.

  In addition, the use of an inexpensive dye D can solve the cost problem of the TaqMan probe.

  FIG. 15 is a schematic diagram showing an embodiment of a probe pair according to the present invention. FIG. 2A shows the first probe 31 constituting the probe pair 3, and FIG. 2B shows the second probe 32.

As shown in FIG. 15A, the first probe 31 is a chemical species that can include other chemical species via a spacer S ₁ in a molecular chain P ₁ that can bind to a predetermined substance. In addition, a first chemical species (host H) having optical activity is bound.

Further, as shown in FIG. 15B, the second probe 32 is included in the host H via the spacer S ₂ and the molecular chain P ₂ that can bind to the same substance as the first probe 31. Dye D, which can be bound.

The molecular chains P ₁ and P ₂ , the host H, and the spacers S ₁ and S ₂ can have the same configuration as that of the probe 1 shown in FIG. However, it is desirable that the sites where the molecular chains P ₁ and P ₂ are bonded in a predetermined substance are not the same or in the vicinity. The dye D may be the same as that described in FIG. 7 and the probe 2 (see FIG. 8).

  FIG. 16 is a schematic diagram showing the behavior of the probe pair 3. (A) shows a state at the time of non-bonding with a predetermined substance, and (B) shows a state at the time of binding.

  When the probe pair 3 is not bonded to a predetermined substance (see symbol T in FIG. 16 (B)), the probe pair 3 is bonded to the probe 32 to the host H bonded to the probe 31 as shown in FIG. 16 (A). The dye D is included.

In contrast, at the time of binding to the substance T shown in FIG. 16B, the distance that the host H of the probe 31 and the dye D of the probe 32 can be included by binding the probe 31 and the probe 32 to the substance T, respectively. Thus, the inclusion state between the host H and the dye D is released. At this time, when the molecular chains P ₁ and P _{2 of} the probe 31 and the probe 32 are bound to the same site or a nearby site of the substance T, the inclusion state of the host H and the dye D is maintained even when the substance T is bound. Therefore, it is desirable that the sites where the molecular chains P ₁ and P ₂ are bonded in a predetermined substance are not the same or in the vicinity.

  As described above, the probe pair 3 is characterized in that the host H and the dye D maintain the inclusion state at the time of non-binding to the substance T, and the inclusion state is released by the binding to the substance T.

  FIG. 17 is a schematic diagram illustrating a method for detecting the binding between the probe pair 3 and the substance T. (A) shows the reaction system before the binding reaction between the probe pair 3 and the substance T, and (B) shows the reaction system after the binding reaction.

Here, a case will be described in which a probe pair 3 composed of molecular chains P ₁ and P ₂ as oligonucleotides (base number n) is used as a primer and a nucleic acid is amplified and detected as a substance T by PCR. Hereinafter, the sense strand of the nucleic acid to be detected is referred to as “sense strand T _s ”, and the antisense strand is referred to as “antisense strand T _a ”.

The molecular chains P ₁ and P ₂ are designed as oligopeptides having base sequences complementary to “sense strand T _s ” and “antisense strand T _a ”, respectively, in the same manner as primers used in normal PCR. It is designed to hybridize to both ends of the amplification target region.

  Prior to the PCR reaction, the probe 31 and the probe 32 maintain the inclusion state of the host H and the dye D as shown in FIG. In this state, Dye D has host H induced chirality.

Therefore, as shown in the figure, when the measurement light L ₁ is irradiated to the dye D included in the host H, the absorbance difference between the left circularly polarized light and the right circularly polarized light is caused by the chirality at the absorption wavelength of the dye D, Measurement target light L ₃ exhibiting circular dichroism is detected.

In PCR reaction shown in FIG. 17 (B), the probe 31 is the sense strand T _s, the probe 32 is connected to the antisense strand T _a, an extension reaction occurs in the drawing direction of the arrow E. At this time, since the probe 31 and the probe 32 are designed to be hybridized to both ends of the amplification target region, the dye D cannot be brought close to the distance that can be included in the host H. The inclusion state with the dye D is released. By this release of the inclusion state, the dye D loses the chirality induced by the host H.

Therefore, even if the measurement light L ₁ is irradiated to the dye D, there is no difference in the absorbance between the left circularly polarized light and the right circularly polarized light absorbed by the dye D, and induced circular dichroism is detected by detecting the measurement target light L _2. It will not be detected.

Thus, according to the probe pair 3 according to the present invention, based on the induced circular dichroism in the measurement target light L _3, to detect the hybridization between the nucleic acid chain and the probe pair 3 is amplified by PCR it can.

In addition, when the sense strand T _s and the antisense strand _Ta are amplified during the PCR process, the amount of probe pair 3 (see FIG. 17A) that maintains the inclusion state of the host H and the dye D decreases. and the amount of probe pairs 3 that bind to the sense strand T _s and antisense strand T _a on behalf becomes increase. Therefore, based on the difference in absorbance of the light to be measured L ₃ (with induced circular dichroism) exhibited by the dye D included in the host H with respect to the left and right circularly polarized light, the nucleic acid strand (sense strand T _s and antisense strand in PCR) It becomes possible to quantitatively analyze the amplification process of T _a ) in real time.

As described above, according to the probe pair 3, based on the induced circular dichroism measurement target light L _3, since it is possible to detect the binding of substance T and the probe pair 3, the conventional intercalator The background fluorescence and noise fluorescence that are generated by the detection method using the fluorescent dye are not generated, and high detection sensitivity can be obtained.

  In addition, the use of an inexpensive dye D can solve the cost problem of the TaqMan probe.

The probe (probe 1 and probe 2, probe pair 3) and the binding detection method using these according to the present invention described above are based on the molecular chain P (P ₁ and P ₂ ) of each probe according to the substance to be detected. Can be used to detect various substances by arbitrarily designing them with a polymer chain having a chemically active group such as oligonucleotides, oligopeptides and polypeptides. Similarly, by arbitrarily designing the molecular chain P of each probe, it can also be used for screening the molecular chain P that can bind to a predetermined substance.

  Among these, the probe and the binding detection method according to the present invention can be particularly preferably used for real-time quantitative detection of an amplified nucleic acid chain in PCR, as described in FIGS. 7, 14, and 17. Compared with methods using fluorescent dyes or TaqMan probes, it is possible to achieve superior detection sensitivity and cost performance.

  Using the probe according to the present invention, binding detection between the oligonucleotide T (base sequence: “5′-GATCGAAGGCGCTACGTTTCGATTC-3 ′”) and the probe was performed under the following conditions.

As the probe, in the probe 1 shown in FIG. 1, the host H is heptas (o-methyl) -γ-cyclodextrin substituent or β-cyclodextrin, the anchor A is lithocholic acid, spacers S ₁ and S ₂ and molecules A probe was used in which the strand P was configured as shown in FIG. The base sequence of the molecular chain P is “5′-GATCGAAACGTAGCGCCTCTCGATC-3 ′” and has a base sequence complementary to the oligonucleotide T.

  The hybridization reaction between this probe and oligonucleotide T is performed in the presence of any one of trimethine cyanine dye (Cy3), neutral red (NR), and rhodamine B (RhB), and the measurement light having a wavelength of 450 nm to 650 nm. The dye was irradiated to measure the circular dichroism in the transmitted light. A 5 × SSC buffer solution prepared so that the final concentrations of probe, dye, and oligonucleotide T were the following concentrations were first annealed at 95 ° C. for 1 minute, and then hybridized for 3 hours while stirring at room temperature. After the reaction, circular dichroism was measured at the same temperature.

  FIG. 18 shows the results of circular dichroism measurement in the presence of a trimethine cyanine dye using a probe in which host H is a heptakis (o-methyl) -γ-cyclodextrin substituent.

The probe concentration was 1.0 × 10 ⁻⁴ M, and the trimethine cyanine dye concentration was 5.0 × 10 ⁻³ M. The concentration of oligonucleotide T was changed stepwise from 0 M, 3.7 × 10 ⁻⁵ M, 6.0 × 10 ⁻⁵ M, and 8.1 × 10 ⁻⁵ M.

FIG. 18A shows the absorption spectrum of the free trimethine cyanine dye, and FIG. 18B shows the measurement result of the circular dichroism spectrum. In (B), the horizontal axis represents the wavelength of transmitted light, and the vertical axis represents circular dichroism (millidegree). The symbols (1) to (4) show the measurement results obtained at oligonucleotide T concentrations of 0 M, 3.7 × 10 ⁻⁵ M, 6.0 × 10 ⁻⁵ M, and 8.1 × 10 ⁻⁵ M, respectively.

  Consistent with the absorption peak of the trimethine cyanine dye (see symbol * in (A)), the maximum value of circular dichroism is observed in (B). (C) is a plot of the maximum circular dichroism values obtained at each concentration of oligonucleotide T. In the figure, the horizontal axis represents the concentration of oligonucleotide T, and the vertical axis represents the maximum value (absolute value) of circular dichroism. Depending on the concentration of oligonucleotide T, an increase in circular dichroism is observed.

  These results indicate that the inclusion state of heptakis (o-methyl) -γ-cyclodextrin substituent and lithocholic acid is released by hybridization of the probe and oligonucleotide T, and trimethine cyanine dye is substituted for lithocholic acid. Is included in the heptakis (o-methyl) -γ-cyclodextrin substituent. The absolute value of circular dichroism induced by the inclusion of this trimethine cyanine dye into the heptakis (o-methyl) -γ-cyclodextrin substituent was enhanced depending on the concentration of oligonucleotide T. Thus, according to the binding detection method of the present invention, it was shown that the oligonucleotide T can be detected quantitatively based on the change width of the circular dichroism.

  FIG. 19 shows the results of measurement of circular dichroism in the presence of neutral red using a probe with host H as a β-cyclodextrin substituent.

The probe concentration was 8.0 × 10 ⁻⁴ M, and the neutral red concentration was 4.0 × 10 ⁻³ M. The concentration of the oligonucleotide T was changed stepwise to 0M, 4.7 × 10 ⁻⁵ M, 5.7 × 10 ⁻⁵ M, and 6.7 × 10 ⁻⁵ M). Other conditions are the same as in FIG.

FIG. 19A shows the absorption spectrum of free neutral red, and FIG. 19B shows the circular dichroism spectrum measurement result. In (B), symbols (1) to (4) indicate the measurement results obtained at oligonucleotide T concentrations of 0 M, 4.7 × 10 ⁻⁵ M, 5.7 × 10 ⁻⁵ M, and 6.7 × 10 ⁻⁵ M, respectively. Show.

  FIG. 20 shows the results of measurement of circular dichroism in the presence of rhodamine B using a probe with host H as a β-cyclodextrin substituent.

The concentration of the probe was 1.2 × 10 ⁻⁴ M, and the concentration of rhodamine B was 6.0 × 10 ⁻³ M. The concentration of oligonucleotide T was changed stepwise from 0M, 5.5 × 10 ⁻⁵ M, 8.0 × 10 ⁻⁵ M, and 10 × 10 ⁻⁵ M. Other conditions are the same as in FIG.

FIG. 20A shows the absorption spectrum of free rhodamine B, and FIG. 20B shows the measurement result of the circular dichroism spectrum. In (B), symbols (1) to (4) indicate the measurement results obtained at oligonucleotide T concentrations of 0M, 5.5 × 10 ⁻⁵ M, 8.0 × 10 ⁻⁵ M, and 10 × 10 ⁻⁵ M, respectively. Show.

  19 and 20, the maximum circular dichroism is observed in accordance with the absorption peak of the dye (see symbol * in FIG. 19A), and the circular dichroism depends on the concentration of oligonucleotide T. (See Figure (C)).

  The probe and probe pair according to the present invention, and the binding detection method using them can be used for detection of nucleic acids, proteins, peptides, etc., and for screening of polymer chains capable of binding to these, and for various diseases. It can be used for elucidating molecular mechanisms and searching for drug discovery targets.

It is a schematic diagram which shows 1st embodiment of the probe which concerns on this invention. FIG. 3 is a diagram showing an example of chemical species that can be suitably employed as the host H. FIG. 4 is a diagram showing an example of chemical species that can be suitably employed as the anchor A. 2 is a schematic diagram showing a specific configuration example of a probe 1. FIG. FIG. 4 is a schematic diagram showing the behavior of the probe 1. 3 is a schematic diagram showing a stem loop structure formed by a molecular chain P. FIG. 6 is a schematic diagram for explaining a method for detecting the binding between the probe 1 and the substance T. FIG. (A) shows the reaction system before the binding reaction between the probe 1 and the substance T, and (B) shows the reaction system after the binding reaction. 2 is a diagram showing an example of chemical species that can be suitably employed as the dye D. FIG. 2 is a schematic diagram showing a specific configuration example of a probe 1. FIG. 10 is a schematic diagram for explaining another method for detecting the binding between the probe 1 and the substance T. FIG. (A) shows the reaction system before the binding reaction between the probe 1 and the substance T, and (B) shows the reaction system after the binding reaction. It is a schematic diagram which shows 2nd embodiment of the probe which concerns on this invention. 3 is a schematic diagram showing a specific configuration example of a probe 2. FIG. 3 is a schematic diagram showing the behavior of a probe 2. FIG. 6 is a schematic diagram for explaining a method for detecting the binding between the probe 2 and the substance T. FIG. (A) shows the reaction system before the binding reaction between the probe 1 and the substance T, and (B) shows the reaction system after the binding reaction. It is a schematic diagram which shows embodiment of the probe pair which concerns on this invention. 4 is a schematic diagram showing the behavior of a probe pair 3. FIG. 4 is a schematic diagram for explaining a method for detecting the binding between the probe pair 3 and the substance T. FIG. (A) shows the reaction system before the binding reaction between the probe pair 3 and the substance T, and (B) shows the reaction system after the binding reaction. It is a figure which shows the result of having measured the circular dichroism in the presence of a trimethine cyanine pigment | dye using the probe which made host H the heptakis (o-methyl) -gamma-cyclodextrin substituent. It is a figure which shows the result of having measured the circular dichroism in the presence of neutral red using the probe which made host H (beta) -cyclodextrin substituent. It is a figure which shows the result of having measured the circular dichroism in the presence of rhodamine B using the probe which made host H (beta) -cyclodextrin substituent.

Explanation of symbols

1, 11, 12, 2, 21, 31, 32 probes
A anchor
B Hydrogen bond
D dye
H host
K substrate surface
L ₁ measuring light
L ₂ and L ₃ light to be measured
P, P ₁ , P ₂ molecular chain
S _1, S _2, S ₃ spacer
St ₁ and St ₂ stem
T substance

Claims (3)

A given substance,
A first chemical species that can include other chemical species in a molecular chain that can bind to the substance and has optical activity, and a second chemical that can be included in the first chemical species A probe bound to a species;
In the presence of a dye that can be included in the first chemical species,
The inclusion state of the first chemical species and the second chemical species maintained when the probe and the substance are not bound is released by the binding of the probe and the substance, thereby the dye. Detecting a binding between the substance and the probe by detecting an induced circular dichroism obtained by inclusion in the first chemical species. The inclusion ability of the dye to the first species, the detection method of claim 1, wherein a lower than said second species of packaging Senno for said first species. The detection method according to claim 1 or 2, which is a real-time PCR method.