Method for detecting target molecule in liquid sample, and kit therefor

Abstract

This method for detecting a target molecule in a liquid sample comprises: a step for mixing the liquid sample with a first hairpin probe that specifically binds to the target molecule, a second hairpin probe that performs a hybridization chain reaction with the first hairpin probe, and a double-stranded nucleic acid sequence-specific fluorescent dye that specifically binds to a specific double-stranded nucleic acid sequence and emits fluorescence, resultantly forming, when the target molecule is present, an amplification product of the first hairpin probe and the second hairpin probe by means of the hybridization chain reaction, and binding the double-stranded nucleic acid sequence-specific fluorescent dye to the specific double-stranded nucleic acid sequence contained in the amplification product; and a step for detecting fluorescence generated by irradiating the double-stranded nucleic acid sequence-specific fluorescent dye with excitation light.

Description

Method for Detecting Target Molecule in Liquid Sample and Kit Therefor

[0001] The present invention relates to a method for detecting a target molecule in a liquid sample and a kit therefor. This application claims priority based on Japanese Patent Application No. 2024-160798 filed in Japan on September 18, 2024, the content of which is incorporated herein by reference.

[0002] Hybridization chain reaction (HCR) is a reaction in which a complementary polymerization reaction between hairpin probes is chained by reacting a target molecule (initiator) and a set of hairpin probes to form an amplification product (see, for example, Patent Document 1 and Non-Patent Document 1).

[0003] Non-Patent Document 1 describes that a hybridization chain reaction targeting a substance such as adenosine triphosphate (ATP) can be performed by introducing an aptamer for a target molecule into a hairpin probe.

[0004] Non-Patent Document 2 describes the application of hybridization chain reaction to the detection of single-stranded DNA fragments floating in a liquid. In Non-Patent Document 2, each of a set of hairpin probes is modified with a fluorescent dye serving as a donor or acceptor, and single-stranded DNA fragments are detected by detecting fluorescence resonance energy transfer (FRET) realized by hybridization chain reaction.

[0005] Incidentally, Non-Patent Document 3 describes that a symmetric cyanine dye derivative having a pyridocyanine skeleton can be used as a DNA fluorescence detection reagent.

[0006] International Publication No. 2021 / 221789

[0007] RM Dirks and NA Pierce, Triggered amplification by hybridization chain reaction, PNAS, vol. 101 (43), 15275-15278, 2004.YS Ang and LYL Yung, Rational design of hybridization chain reaction monomers for robust signal amplification, Chem. Commun., 52, 4219-4222, 2016.K. Uno, et al., N-aryl pyrido cyanine derivatives are nuclear and organelle DNA markers for two-photon and super-resolution imaging, Nat Commun., 12 (1), 2650, 2021.

[0008] There is a need for technologies to detect target molecules in liquid samples. For example, the detection of miRNA (microRNA) has been reported to be useful in cancer diagnosis, radiation exposure diagnosis, and artificial insemination embryo diagnosis. Traditionally, miRNA has been detected using RT-PCR or microarrays. However, these methods are time-consuming and costly, and there are problems such as sample loss during miRNA extraction.

[0009] According to the method described in Non-Patent Document 2, single-stranded DNA fragments suspended in a liquid can be detected by hybridization chain reaction. However, the method described in Non-Patent Document 2 has several drawbacks, including the possibility that the hybridization chain reaction may be inhibited by the fluorescence modification of the hairpin probe, and that the signal-to-noise ratio may decrease due to the presence of unreacted hairpin probes.

[0010] The present invention aims to provide an improved technique for detecting target molecules in a liquid sample.

[0011] The present invention includes the following aspects: [1] A method for detecting a target molecule in a liquid sample, comprising the steps of: mixing the liquid sample with a first hairpin probe that specifically binds to the target molecule, a second hairpin probe that performs a hybridization chain reaction with the first hairpin probe, and a double-stranded nucleic acid sequence-specific fluorescent dye that specifically binds to a specific double-stranded nucleic acid sequence and emits fluorescence, wherein if the target molecule is present, amplified products of the first hairpin probe and the second hairpin probe are formed by the hybridization chain reaction, and the double-stranded nucleic acid sequence-specific fluorescent dye binds to the specific double-stranded nucleic acid sequence contained in the amplified products; and irradiating the double-stranded nucleic acid sequence-specific fluorescent dye with excitation light to detect the fluorescence generated. [2] The method according to [1], wherein the target molecule is a single-stranded nucleic acid or a substance that specifically binds to a nucleic acid aptamer. [3] A kit for detecting a target molecule in a liquid sample, comprising: a first hairpin probe that specifically binds to the target molecule; a second hairpin probe that performs a hybridization chain reaction with the first hairpin probe; and a double-stranded nucleic acid sequence-specific fluorescent dye that specifically binds to a specific double-stranded nucleic acid sequence and emits fluorescence. [4] The kit according to [3], wherein the target molecule is a single-stranded nucleic acid or a substance that specifically binds to a nucleic acid aptamer.

[0012] According to the present invention, an improved technique for detecting target molecules in a liquid sample can be provided.

[0013] Figure 1 is a schematic diagram illustrating an example of a typical hybridization chain reaction. Figure 2 is a schematic diagram illustrating an example of a method for detecting amplified products formed by a hybridization chain reaction. Figure 3 is a schematic diagram illustrating one embodiment of a method for detecting target molecules in a liquid sample. Figure 4 is a graph showing the fluorescence intensity measured in Experimental Example 1. Figure 5 is a photograph showing the results of native gel electrophoresis in Experimental Example 1. The upper part of Figure 6 shows the amplified products formed by the hybridization chain reaction when using the first hairpin probe, the second hairpin probe, and the third hairpin probe. The middle part of Figure 6 shows the amplified products formed by the hybridization chain reaction when using the first hairpin probe and the second hairpin probe. The lower part of Figure 6 shows an experimental system for detecting the amplified products formed by the hybridization chain reaction when using the first hairpin probe, the second hairpin probe, and the third hairpin probe using FRET. The upper part of Figure 7 is a graph showing the fluorescence intensity measured in Experimental Example 2. The lower part of Figure 7 is a magnified view of the low-concentration region of the target molecule in the graph in the upper part of Figure 7. The upper and lower parts of Figure 8 are photographs showing the results of native gel electrophoresis in Experimental Example 2. The upper part of Figure 9 shows the hairpin DNA used in Experimental Example 3. The middle and lower parts of Figure 9 are graphs showing the fluorescence intensity measured in Experimental Example 3. The upper and lower parts of Figure 10 are photographs showing the results of native gel electrophoresis in Experimental Example 4. The upper, middle, and lower parts of Figure 11 are graphs showing the results of detecting the amplified product formed by the hybridization chain reaction by fluorescence emission in Experimental Example 4.

[0014] First, let's explain hybridization chain reactions. Figure 1 is a schematic diagram illustrating a typical example of a hybridization chain reaction. In the example in Figure 1, the target molecule has the base sequence represented by "5'-a'b'-3'". Hairpin probe 1 has the base sequence represented by "5'-b'cba-3'". Hairpin probe 2 has the base sequence represented by "5'-c'ba'b'-3'". Here, "a" and "a'", "b" and "b'", and "c" and "c'" represent complementary base sequences.

[0015] When the target molecule is absent, hairpin probe 1 and hairpin probe 2 remain stable. However, as shown in Figure 1, when the target molecule is present, first, the "a'" region of the target molecule and the "a" region of hairpin probe 1 hybridize. Subsequently, while the hairpin of hairpin probe 1 is cleaved, the "b'" region of the target molecule and the "b" region of hairpin probe 1 hybridize. As a result, the "cb'" region of hairpin probe 1 is exposed.

[0016] Next, the "c'" region of hairpin probe 2 hybridizes with the "c" region of hairpin probe 1. Subsequently, the "b'" region of hairpin probe 1 and the "b" region of hairpin probe 2 hybridize while the hairpin of hairpin probe 2 is cleaved. As a result, the "a'b'" region of hairpin probe 2 is exposed.

[0017] Next, the exposed "a'" region of hairpin probe 2 hybridizes with the "a" region of hairpin probe 1. Subsequently, the hairpin of hairpin probe 1 is cleaved, and the "b'" region of hairpin probe 2 hybridizes with the "b" region of hairpin probe 2. As a result, the "cb'" region of hairpin probe 1 is exposed.

[0018] Subsequently, the same process is repeated to form amplified products of hairpin probe 1 and hairpin probe 2. The formation of amplified products indicates the presence of the target molecule.

[0019] Conventionally, the amplification products of hairpin probe 1 and hairpin probe 2 have been detected by methods such as native gel electrophoresis (see, for example, Non-Patent Document 1), detection of deposited catalyst reporters (see, for example, Patent Document 1), and detection of FRET (see, for example, Non-Patent Document 2).

[0020] Figure 2 is a schematic diagram illustrating an example of a method for detecting the amplification products of hairpin probe 1 and hairpin probe 2. In the example in Figure 2, the hybridization chain reaction is designed so that a portion of hairpin probe 1 and hairpin probe 2 is ejected from the amplification product. A labeled probe having a complementary base sequence is hybridized to this portion, and the amplification product is detected by detecting the accumulation of the labeled probe using a dye or enzyme labeled on the labeled probe. In the method in Figure 2, the presence of unbound labeled probes becomes background noise, so it is necessary to wash and remove them.

[0021] [Method for detecting target molecules in a liquid sample] In one embodiment, the present invention provides a method for detecting target molecules in a liquid sample, comprising the steps of: mixing a first hairpin probe that specifically binds to the target molecule, a second hairpin probe that performs a hybridization chain reaction with the first hairpin probe, and a double-stranded nucleic acid sequence-specific fluorescent dye that specifically binds to a specific double-stranded nucleic acid sequence and emits fluorescence with the liquid sample; if the target molecule is present, amplified products of the first hairpin probe and the second hairpin probe are formed by the hybridization chain reaction, and the double-stranded nucleic acid sequence-specific fluorescent dye binds to the specific double-stranded nucleic acid sequence contained in the amplified products; and irradiating the double-stranded nucleic acid sequence-specific fluorescent dye with excitation light to detect the fluorescence generated.

[0022] The liquid sample is not particularly limited and can include, for example, serum, plasma, urine, saliva, cerebrospinal fluid, etc. Target molecules include disease biomarkers, and specifically, substances that specifically bind to single-stranded nucleic acids and nucleic acid aptamers. Target molecules will be discussed later.

[0023] The method of this embodiment uses a double-stranded nucleic acid sequence-specific fluorescent dye to detect the amplification products of the first and second hairpin probes. As will be described later in the examples, the method of this embodiment significantly improves the signal-to-noise ratio and detection sensitivity compared to conventional methods that involve fluorescently modifying hairpin probes to detect FRET.

[0024] Double-stranded nucleic acid sequence-specific fluorescent dyes specifically bind to double-stranded nucleic acids that have a particular base sequence. When these fluorescent dyes bind to double-stranded nucleic acids, the fluorescence emission efficiency generated by irradiation with excitation light is significantly increased.

[0025] The double-stranded nucleic acid sequence recognized by the double-stranded nucleic acid sequence-specific fluorescent dye is not present when the first and second hairpin probes are forming hairpins, but rather when the first and second hairpin probes form amplified products through a hybridization chain reaction. The first and second hairpin probes should be designed in this manner.

[0026] The double-stranded nucleic acid sequence is not particularly limited, but for example, the double-stranded DNA sequence shown in formula (1) below can be used as an example. In this case, the double-stranded nucleic acid sequence-specific fluorescent dye specifically binds to the double-stranded DNA sequence shown in formula (1) below, which is contained in the amplification product formed by the hybridization chain reaction.

[0027]

[0028] Examples of fluorescent dyes that specifically bind to the double-stranded DNA sequence shown in formula (1) above include 4',6'-diamidino-2-phenylindole (DAPI) and the compound shown in formula (2) below. The compound shown in formula (2) below belongs to a group of fluorescent dyes called Kakshine.

[0029] [In formula (2), R １ and R ２ Each of these is independently either absent, or a methoxy group, a dimethylamino group, or a diethylamino group, X －This is a chloride ion or a p-toluenesulfonate anion.

[0030] DAPI and the compound represented by formula (2) above specifically bind to the double-stranded DNA sequence represented by formula (1) above, and when bound to the double-stranded DNA, the fluorescence emission efficiency generated by irradiation with excitation light is greatly increased.

[0031] The double-stranded DNA sequence shown in formula (1) above is a sequence consisting of 4 base pairs, but the length of the double-stranded nucleic acid sequence is not limited to this, and may consist of, for example, 2 to 20 base pairs.

[0032] Figure 3 is a schematic diagram illustrating an example of the method of this embodiment. For example, in the example in Figure 3, the base sequence 310 of one strand of a double-stranded nucleic acid sequence is introduced into the hairpin loop region of the first hairpin probe HP1, and a base sequence 310' complementary to base sequence 310 is introduced into the single-stranded portion of the second hairpin probe HP2. As a result, the double-stranded nucleic acid sequence recognized by the double-stranded nucleic acid sequence-specific fluorescent dye 320 (the double-stranded nucleic acid sequence formed by the hybridization of base sequences 310 and 310') does not exist when the first hairpin probe HP1 and the second hairpin probe HP2 are forming hairpins, but is formed when the first hairpin probe HP1 and the second hairpin probe HP2 form amplification products through a hybridization chain reaction.

[0033] In Figure 3, the process by which the first hairpin probe HP1 and the second hairpin probe HP2 form amplified products via a hybridization chain reaction when a target molecule is present is the same as that described in Figure 1.

[0034] The double-stranded nucleic acid sequence-specific fluorescent dye 320 specifically recognizes and binds to the double-stranded nucleic acid sequence in which base sequences 310 and 310' have hybridized, among the amplification products formed by the hybridization chain reaction.

[0035] When excitation light is shone on a double-stranded nucleic acid sequence-specific fluorescent dye 320' bound to a double-stranded nucleic acid, specific fluorescence is generated. The detection of this fluorescence indicates the presence of the target molecule in the sample.

[0036] According to the method of this embodiment, there is no need to fluorescently modify the hairpin probe. Therefore, the hybridization chain reaction is not inhibited by fluorescent modification of the hairpin probe. Furthermore, according to the method of this embodiment, fluorescent dyes that are not bound to double-stranded nucleic acids do not emit fluorescence, and only fluorescent dyes that are specifically bound to the double-stranded nucleic acid sequence emit fluorescence. Therefore, unlike conventional methods for detecting FRET, the signal-to-noise ratio does not decrease due to the presence of unreacted hairpin probes.

[0037] In the example shown in Figure 3, the base sequence 310 of one strand of a double-stranded nucleic acid sequence was introduced into the hairpin loop region of the first hairpin probe HP1, and a base sequence 310' complementary to base sequence 310 was introduced into the single-stranded portion of the second hairpin probe HP2. However, the position of the double-stranded nucleic acid sequence to which the double-stranded nucleic acid sequence-specific fluorescent dye 320 specifically binds is not limited to this.

[0038] For example, as shown in the example in Figure 2, the hybridization chain reaction may be designed so that a portion of hairpin probe 1 and hairpin probe 2 are ejected from the amplified product, and a probe having a complementary base sequence is hybridized to this portion, thereby forming a double-stranded nucleic acid sequence to which the double-stranded nucleic acid sequence-specific fluorescent dye 320 specifically binds.

[0039] Furthermore, as will be described later in the examples, a third hairpin probe may be used in addition to the first and second hairpin probes. This makes it possible to design a hairpin probe in which the region in the amplification product to which the double-stranded nucleic acid sequence-specific fluorescent dye binds is approximately twice as large compared to when the first and second hairpin probes are used. This further improves the detection sensitivity of the target molecule.

[0040] The hairpin probe may be formed of a nucleic acid analog at least in part. Examples of the nucleic acid analog include, for example, Locked Nucleic Acid (LNA), Peptide Nucleic Acid (PNA), Bridged Nucleic Acid (BNA), Morpholino phosphoramidate, and the like.

[0041] For example, when the double-stranded nucleic acid sequence-specific fluorescent dye used does not bind to the nucleic acid analog, a region in the base sequence of the hairpin probe where the double-stranded nucleic acid sequence-specific fluorescent dye is not desired to bind can be formed of the nucleic acid analog. Thereby, the degree of freedom in the design of the hairpin probe can be improved.

[0042] In the method of the present embodiment, the target molecule may be a single-stranded nucleic acid or a substance that specifically binds to a nucleic acid aptamer.

[0043] Examples of the single-stranded nucleic acid include mRNA, miRNA, environmental RNA, circulating tumor DNA (ctDNA), and the like.

[0044] The first hairpin probe does not need to hybridize to the entire single-stranded nucleic acid that is the target molecule, and as long as it can cleave the hairpin, it can start a hybridization chain reaction by hybridizing to a part of the single-stranded nucleic acid. Therefore, the length of the single-stranded nucleic acid serving as the target molecule is not particularly limited.

[0045] Examples of the substance that specifically binds to the nucleic acid aptamer include proteins, metabolites, artificial compounds, bacteria such as Escherichia coli, artificial nanoparticles, and the like.

[0046] By making a part of the hairpin probe have the base sequence of the nucleic acid aptamer, when the hairpin probe binds to a substance, a hybridization chain reaction can proceed to form an amplification product.

[0047] The first hairpin probe does not need to bind to the entire target molecule; as long as it can cleave the hairpin, it can initiate a hybridization chain reaction by hybridizing with a portion of the substance. Therefore, the size (molecular weight, etc.) of the target molecule is not particularly limited.

[0048] [Kit for detecting target molecules in liquid samples] In one embodiment, the present invention provides a kit for detecting target molecules in a liquid sample, comprising: a first hairpin probe that specifically binds to the target molecule; a second hairpin probe that performs a hybridization chain reaction with the first hairpin probe; and a double-stranded nucleic acid sequence-specific fluorescent dye that specifically binds to a specific double-stranded nucleic acid sequence and emits fluorescence.

[0049] The kit of this embodiment allows for the suitable implementation of the method described above for detecting target molecules in a liquid sample.

[0050] In the kit of this embodiment, the liquid sample, target molecule, first hairpin probe, second hairpin probe, double-stranded nucleic acid sequence-specific fluorescent dye, etc., are the same as those described above. The target molecule may be a single-stranded nucleic acid or a substance that specifically binds to a nucleic acid aptamer.

[0051] As described above, the kit of this embodiment may further include a third hairpin probe in addition to the first and second hairpin probes.

[0052] The kit of this embodiment may be designed, for example, as described above in Figure 2, so that as a result of the hybridization chain reaction, a portion of hairpin probe 1 and hairpin probe 2 are ejected from the amplified product, and a probe having a complementary base sequence to this portion is hybridized, thereby forming a double-stranded nucleic acid sequence to which a double-stranded nucleic acid sequence-specific fluorescent dye specifically binds. In this case, the kit of this embodiment may further include a probe having a complementary base sequence to the single-stranded region ejected from the amplified product.

[0053] The present invention will now be described in more detail with reference to examples, but the present invention is not limited to the following examples.

[0054] [Experimental Example 1] (Detection of Target Molecules 1) Target molecules were detected using a hybridization chain reaction. In a 96-well plate, the target molecule, a first hairpin probe, a second hairpin probe, and a double-stranded nucleic acid sequence-specific fluorescent dye were mixed and a hybridization chain reaction was carried out to detect the target molecule.

[0055] As the target molecule, a DNA fragment having the base sequence 5'-GCACGTGGAGCGAGATCAGAA-3' (SEQ ID NO: 1) was used. As the first hairpin probe, a DNA fragment having the base sequence 5'-TTCTGATCTCGCTCCACGTGCTCGAATTCTGCACGTGGAGCG-3' (SEQ ID NO: 2) was used. As the second hairpin probe, a DNA fragment having the base sequence 5'-GCACGTGGAGCGAGATCAGAACGCTCCACGTGCAGAATTCGA-3' (SEQ ID NO: 3) was used. As the double-stranded nucleic acid sequence-specific fluorescent dye, the compound shown in formula (3) below was used. The compound shown in formula (3) below is a type of fluorescent dye called Kakshine.

[0056] [In formula (3), X － This is a chloride ion.

[0057] To a 1× SSC buffer (150 mM NaCl, 15 mM sodium citrate) containing 0.1% Tween 20, a 100 nM first hairpin probe, a 100 nM second hairpin probe, and target molecules at concentrations of 0, 12.5, 25, 125, 250, 1,250, and 2,500 pM were mixed. Subsequently, the mixtures were allowed to stand at room temperature (20–28°C) for 2 hours to allow the hybridization chain reaction to proceed.

[0058] Subsequently, 200 nM of the Kakshine fluorescent dye shown in formula (3) above and 0.02 mM of the anti-fading agent Trolox were added, and excitation light at a wavelength of 532 nm was irradiated to detect fluorescence at a wavelength of 546 nm. Figure 4 is a graph showing the measured fluorescence intensity. A magnified graph of the low-concentration region of the target molecule is shown in the lower right of Figure 4. As a result, fluorescence corresponding to the concentration of the target molecule was detected. Furthermore, when the same measurement was performed with greater precision, it was shown that the detection limit for the target molecule is 20 pM.

[0059] Figure 5 shows photographs of the results of native gel electrophoresis performed on each sample after the hybridization chain reaction, with the addition of the DNA staining dye Gel Red (Biotium). The results confirmed that as the concentration of the target molecule increased, the consumption of the hairpin probe increased, leading to an increase in the amount of amplified product formed.

[0060] [Experimental Example 2] (Detection of Target Molecules 2) miR744, known as a pancreatic cancer marker, was used as the target molecule and detected using a hybridization chain reaction.

[0061] As the target molecule, a chemically synthesized RNA fragment having the base sequence miR744 (hsa-miR-744-5p, 5'-UGCGGGGCUAGGGCUAACAGCA-3', SEQ ID NO: 4) was used. As the first hairpin probe, a DNA fragment having the base sequence 5'-ACGAATTGATGCTGTTAGCCCACGAATTGAGGGCTAACAGCA-3' (SEQ ID NO: 5) was used. As the second hairpin probe, a DNA fragment having the base sequence 5'-GGGCTAACAGCATCAATTCGTTGCTGTTAGCCCTCAATTCGT-3' (SEQ ID NO: 6) was used. In addition to the first and second hairpin probes, a third hairpin probe (5'-GGGCTAACAGCATCAATTCGTTGCTGTTAGCCCTAGCCCCGCA-3', SEQ ID NO: 7) was also used in this experiment. As the double-stranded nucleic acid sequence-specific fluorescent dye, the Kakshine fluorescent dye shown in formula (3) above was used.

[0062] For comparison, a detection system that detects amplified products using FRET was also investigated. Specifically, as the first hairpin probe, the hairpin probe of SEQ ID NO: 5 described above was modified with a donor fluorescent dye (ATTO550, ATTO-TEC) (5'-ATTO550-C6-ACGAATTGATGCTGTTAGCCCACGAATTGAGGGCTAACAGCA-3', SEQ ID NO: 8). As the second hairpin probe, the hairpin probe of SEQ ID NO: 6 described above was modified with an acceptor fluorescent dye (Alexa647, Thermo Fisher Scientific) (5'-Alexa647-C6-GGGCTAACAGCATCAATTCGTTGCTGTTAGCCCTCAATTCGT-3', SEQ ID NO: 9). In addition to the first and second hairpin probes, a third hairpin probe (5'-GGGCTAACAGCATCAATTCGTTGCTGTTAGCCCTAGCCCCGCA-3', Sequence ID 7) was also used.

[0063] The upper part of Figure 6 shows the amplified products formed by the hybridization chain reaction when using the first hairpin probe (SEQ ID NO: 5, indicated as "HP1"), the second hairpin probe (SEQ ID NO: 6, indicated as "HP2"), and the third hairpin probe (SEQ ID NO: 7, indicated as "HP3").

[0064] The lower panel of Figure 6 shows an experimental setup for detecting amplified products formed by hybridization chain reactions using a first hairpin probe (sequence number 8, indicated as "HP1'"), a second hairpin probe (sequence number 9, indicated as "HP2'"), and a third hairpin probe (sequence number 7, indicated as "HP3"), using FRET. In the lower panel of Figure 6, "D" indicates the donor fluorescent dye (ATTO550), and "A" indicates the acceptor fluorescent dye (Alexa647).

[0065] The middle section of Figure 6 shows the amplified products formed by the hybridization chain reaction when the first hairpin probe (5'-ACGAATTGATGCTGTTAGCCCTGCGGGGCTAGGGCTAACAGCA-3', SEQ ID NO: 10, indicated as "HP1") and the second hairpin probe (5'-GGGCTAACAGCATCAATTCGTTGCTGTTAGCCCTAGCCCCGCA-3', SEQ ID NO: 11, indicated as "HP2") are used, without using the third hairpin probe.

[0066] As shown in the upper and middle panels of Figure 6, when using the third hairpin probe (upper panel of Figure 6), it is possible to design a hairpin probe that approximately doubles the region in the amplification product to which the double-stranded nucleic acid sequence-specific fluorescent dye binds compared to when the third hairpin probe is not used (middle panel of Figure 6). This further improves the detection sensitivity of the target molecule.

[0067] When using the Kakshine fluorescent dye shown in formula (3) above, a 25 nM first hairpin probe, a 25 nM second hairpin probe, and a 25 nM third hairpin probe, along with target molecules at concentrations of 0, 5, 10, 25, 50, 100, 500, and 1,000 pM respectively, were mixed in 1× SSC buffer (150 mM NaCl, 15 mM sodium citrate) containing 0.1% Tween 20. The mixture was then allowed to stand at room temperature (20-28°C) for 2 hours to allow the hybridization chain reaction to proceed. Subsequently, 200 nM of the Kakshine fluorescent dye shown in formula (3) above and 0.02 mM of the anti-fading agent Trolox were added, and the mixture was irradiated with excitation light at a wavelength of 532 nm to detect fluorescence at a wavelength of 546 nm.

[0068] When using FRET, a 1× SSC buffer (150 mM NaCl, 15 mM sodium citrate) containing 0.1% Tween 20 was mixed with a 25 nM first hairpin probe, a 25 nM second hairpin probe, a 25 nM third hairpin probe, and target molecules at concentrations of 0, 5, 10, 25, 50, 100, 500, and 1,000 pM, respectively. Subsequently, the mixture was allowed to stand at room temperature (20–28°C) for 2 hours to allow the hybridization chain reaction to proceed. After that, 0.02 mM Trolox, a colorfastness inhibitor, was added, and light with a wavelength of 532 nm, which is the excitation light for the donor fluorescent dye (ATTO550), was irradiated. Light with a wavelength of 670 nm, originating from the emission of the acceptor fluorescent dye (Alexa647), was detected.

[0069] The upper part of Figure 7 is a graph showing the measured fluorescence intensity. The lower part of Figure 7 is a graph magnified over the low-concentration region of the target molecule. In Figure 7, "Kakshine" indicates the result of detecting the amplified product using the Kakshine fluorescent dye shown in formula (3) above, and "FRET" indicates the result of detecting the amplified product using FRET. The vertical axis of the graph shows the signal / background ratio.

[0070] In the detection of amplified products using Kakshine fluorescent dyes, the signal / background ratio was calculated using the following formula (F1). In formula (F1), the negative control refers to the sample without the target molecule (0 pM). Signal / background ratio = Fluorescence intensity at 546 nm / Fluorescence intensity at 546 nm in the negative control…(F1)

[0071] In the detection of amplified products by FRET, the signal / background ratio was calculated using the following formula (F2). In formula (F2), the negative control refers to the sample without the target molecule (0 pM). Signal / background ratio = [fluorescence intensity at 670 nm / fluorescence intensity at 580 nm] / [fluorescence intensity at 670 nm in the negative control / fluorescence intensity at 580 nm in the negative control] ... (F2)

[0072] As a result, fluorescence corresponding to the concentration of the target molecule was detected in both cases: when the amplified product was detected using Kakshine fluorescent dye and when the amplified product was detected using FRET. However, the signal-to-background ratio was significantly higher when the amplified product was detected using Kakshine fluorescent dye. Furthermore, when the amplified product was detected using Kakshine fluorescent dye, the detection limit for the target molecule was shown to be 10 pM.

[0073] The upper panel of Figure 8 shows the results of native gel electrophoresis after adding the DNA staining dye Gel Red (Biotium) to each sample after the hybridization chain reaction when amplification products were detected using Kakshine fluorescent dye. The lower panel of Figure 8 shows the results of native gel electrophoresis after adding the DNA staining dye Gel Red to each sample after the hybridization chain reaction when amplification products were detected using FRET. As a result, it was confirmed that the amount of amplification product formed increased as the concentration of the target molecule increased. However, the amount of amplification product was less than in the upper panel. This was thought to be because hybridization was inhibited by the fluorescent dye modified at the end of the hairpin probe.

[0074] [Experimental Example 3] (Investigation of Nucleic Acid Analogues) The hairpin DNA shown in the upper panel of Figure 9 (5'-CGCGAATTCGCGTTTTCGCGAATTCGCG-3', Sequence ID No. 18) was used for the investigation. The hairpin DNA of Sequence ID No. 18 has a binding sequence for Kakshine fluorescent dye (the double-stranded DNA sequence shown in formula (1) above) in the stem portion (double-stranded portion).

[0075] First, hairpin DNA of sequence number 18 was synthesized using native nucleic acid. Next, 200 nM of Kakshine fluorescent dye shown in formula (3) above and 0.02 mM of the anti-fading agent Trolox were added to serially diluted hairpin DNA, and excitation light at a wavelength of 532 nm was irradiated, and fluorescence at a wavelength of 546 nm was detected. The middle panel of Figure 9 is a graph showing the measured fluorescence intensity. As a result, fluorescence corresponding to the concentration of hairpin DNA was detected. This result indicates that hairpin DNA with a Kakshine fluorescent dye binding sequence in the stem region cannot be used in a hybridization chain reaction detected by Kakshine fluorescent dye.

[0076] Next, hairpin DNA (SEQ ID NO: 19) was synthesized by replacing the 5'-AATT-3' base sequence portion of the hairpin DNA of SEQ ID NO: 18 with a nucleic acid analog (LNA). Subsequently, 200 nM of Kakshine fluorescent dye shown in formula (3) above and 0.02 mM of the anti-fading agent Trolox were added to serially diluted hairpin DNA, and the samples were irradiated with excitation light at a wavelength of 532 nm, and fluorescence at a wavelength of 546 nm was detected. The lower part of Figure 9 is a graph showing the measured fluorescence intensity. As a result, it became clear that no fluorescence of the Kakshine fluorescent dye was detected in the hairpin DNA of SEQ ID NO: 19. This result indicates that when the presence of a Kakshine fluorescent dye binding sequence in the stem portion of a hairpin probe is unavoidable, it is possible to use it in a hybridization chain reaction detected by the Kakshine fluorescent dye by replacing that portion with a nucleic acid analog.

[0077] [Experimental Example 4] (Investigation of fluorescent dyes other than Kakshine) As a fluorescent dye other than Kakshine, we investigated the detection of hybridization chain reactions using 4',6'-diamidino-2-phenylindole (DAPI).

[0078] DAPI is a double-stranded nucleic acid sequence-specific fluorescent dye that binds to the double-stranded DNA sequence shown in formula (1) above (hereinafter sometimes referred to as the "AATT sequence"). The binding sequence of DAPI is the same as that of the Kakshine fluorescent dye. For comparison, we also conducted studies using SYBR Gold (Thermo Fisher Scientific), a fluorescent dye that binds to double-stranded DNA in a sequence-nonspecific manner and emits fluorescence.

[0079] First, amplified hybridization chain reaction products containing the AATT sequence and amplified hybridization chain reaction products without the AATT sequence were prepared.

[0080] In the preparation of the amplified hybridization chain reaction product containing the AATT sequence, a DNA fragment having the nucleotide sequence 5'-GCACGTGGAGCGAGATCAGAA-3' (SEQ ID NO: 1) was used as the target molecule. Furthermore, a DNA fragment having the nucleotide sequence 5'-TTCTGATCTCGCTCCACGTGCTCGAATTCTGCACGTGGAGCG-3' (SEQ ID NO: 2) was used as the first hairpin probe. Additionally, a DNA fragment having the nucleotide sequence 5'-GCACGTGGAGCGAGATCAGAACGCTCCACGTGCAGAATTCGA-3' (SEQ ID NO: 3) was used as the second hairpin probe.

[0081] In the preparation of amplified hybridization chain reaction products that do not contain the AATT sequence, a DNA fragment having the nucleotide sequence 5'-CGGTCGGTGAGCATCTTCCAT-3' (SEQ ID NO: 20) was used as the target molecule. In addition, a DNA fragment having the nucleotide sequence 5'-ATGGAAGATGCTCACCGACCGTTCATGCAACGGTCGGTGAGC-3' (SEQ ID NO: 21) was used as the first hairpin probe. In addition, a DNA fragment having the nucleotide sequence 5'-CGGTCGGTGAGCATCTTCCATGCTCACCGACCGTTGCATGAA-3' (SEQ ID NO: 22) was used as the second hairpin probe.

[0082] A 100 nM first hairpin probe, a 100 nM second hairpin probe, and target molecules of 0, 1.25, 2.5, 5, 10, and 20 nM were mixed into a 1× SSC buffer (150 mM NaCl, 15 mM sodium citrate) containing 0.1% Tween 20. Subsequently, the mixtures were allowed to stand at room temperature (20–28°C) for 2 hours to allow the hybridization chain reaction to proceed.

[0083] Figure 10 is a photograph showing the results of native gel electrophoresis performed on each sample after the hybridization chain reaction, with the addition of the DNA staining dye Gel Red (Biotium). The upper part of Figure 10 shows the amplification products of the hybridization chain reaction containing the AATT sequence, and the lower part of Figure 10 shows the amplification products of the hybridization chain reaction without the AATT sequence.

[0084] As a result, in all samples, it was confirmed that as the concentration of the target molecule increased, the consumption of the hairpin probe increased, and at a target molecule concentration of 20 nM, all of the hairpin probe was used for hybridization, with no unpolymerized hairpin probe remaining.

[0085] The upper part of Figure 11 is a graph showing the results of adding 200 nM of the Kakshine fluorescent dye shown in formula (3) above and 0.02 mM of the anti-fading agent Trolox to each sample after the hybridization chain reaction, and then irradiating it with excitation light at a wavelength of 532 nm and detecting fluorescence at a wavelength of 546 nm. As a result, fluorescence corresponding to the concentration of the target molecule was detected in the amplified product of the hybridization chain reaction containing the AATT sequence. On the other hand, fluorescence corresponding to the concentration of the target molecule was not detected in the amplified product of the hybridization chain reaction without the AATT sequence.

[0086] The middle section of Figure 11 shows a graph illustrating the results of adding 200 nM DAPI and 0.02 mM Trolox (a fluorescence inhibitor) to each sample after a hybridization chain reaction, irradiating it with excitation light at a wavelength of 358 nm, and detecting fluorescence at a wavelength of 461 nm. The results showed that in the amplified product of the hybridization chain reaction containing the AATT sequence, fluorescence corresponding to the concentration of the target molecule was detected. On the other hand, in the amplified product of the hybridization chain reaction without the AATT sequence, fluorescence corresponding to the concentration of the target molecule was not detected. This result indicates that even double-stranded nucleic acid sequence-specific fluorescent dyes other than Kakshine fluorescent dyes can be used to detect hybridization chain reactions using the same principle.

[0087] The lower panel of Figure 11 shows a graph of the results obtained when each sample after the hybridization chain reaction was treated with 200 nM SYBR Gold and 0.02 mM Trolox (a chromogenic inhibitor), irradiated with excitation light at a wavelength of 495 nm, and fluorescence at a wavelength of 540 nm was detected. As a result, regardless of whether the amplified product of the hybridization chain reaction contained an AATT sequence or not, no fluorescence corresponding to the concentration of the target molecule was detected. Furthermore, since the hairpin probe before the hybridization chain reaction also forms double-stranded DNA, it is thought that no change in fluorescence intensity was observed before and after the hybridization chain reaction when SYBR Gold was used.

[0088] According to the present invention, an improved technique for detecting target molecules in a liquid sample can be provided.

[0089] HP1...First hairpin probe, HP2...Second hairpin probe, HP3...Third hairpin probe, 310,310'...Base sequence, 320...Double-stranded nucleic acid sequence-specific fluorescent dye.

Claims

1. A method for detecting a target molecule in a liquid sample, comprising: mixing a first hairpin probe that specifically binds to the target molecule, a second hairpin probe that performs a hybridization chain reaction with the first hairpin probe, and a double-stranded nucleic acid sequence-specific fluorescent dye that specifically binds to a specific double-stranded nucleic acid sequence and emits fluorescence into the liquid sample; wherein, if the target molecule is present, amplified products of the first hairpin probe and the second hairpin probe are formed by the hybridization chain reaction, and the double-stranded nucleic acid sequence-specific fluorescent dye binds to the specific double-stranded nucleic acid sequence contained in the amplified products; and detecting fluorescence generated by irradiating the double-stranded nucleic acid sequence-specific fluorescent dye with excitation light.

2. The method according to claim 1, wherein the target molecule is a single-stranded nucleic acid or a substance that specifically binds to a nucleic acid aptamer.

3. A kit for detecting a target molecule in a liquid sample, comprising: a first hairpin probe that specifically binds to the target molecule; a second hairpin probe that performs a hybridization chain reaction with the first hairpin probe; and a double-stranded nucleic acid sequence-specific fluorescent dye that specifically binds to a specific double-stranded nucleic acid sequence and emits fluorescence.

4. The kit according to claim 3, wherein the target molecule is a single-stranded nucleic acid or a substance that specifically binds to a nucleic acid aptamer.

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