Methods for detecting target nucleic acids

Abstract

The present application relates to a method for detecting a target nucleic acid, comprising: a step of cleaving a first wing of a first split structure formed by a target nucleic acid, a first nucleic acid, and a second nucleic acid; a step of cleaving a second wing of a second split structure formed by a third nucleic acid, the cleaved first wing, and a fourth nucleic acid; a step of detecting the presence of the target nucleic acid by detecting the cleaved second wing; wherein the step of cleaving the first wing and the step of cleaving the second wing are performed by cleaving the first wing and the second wing with a wing endonuclease having an amino acid sequence having a sequence homology of 96% or more with an amino acid sequence of a wing endonuclease of Thermococcus kodakarensis KOD1 strain or the like.

Description

Technical Field

[0001] This invention relates to a method for detecting target nucleic acids. Background Technology

[0002] Various diagnostic methods based on the detection of target nucleic acids are known. Examples of such methods include constitution diagnosis based on single nucleotide polymorphism (SNP) analysis, predictive diagnosis of the efficacy and side effects of drugs (e.g., anticancer agents) based on somatic mutation analysis, and diagnosis of infectious diseases based on the detection of viral DNA or RNA. In these diagnostic applications, the presence of target nucleic acids is often relatively low, thus requiring methods capable of highly accurate quantitative analysis of target nucleic acids. Digital measurement methods are known, for example, for highly accurate quantitative analysis of target nucleic acids.

[0003] Digital measurement is a quantitative method that involves diluting target nucleic acids or other biomolecules to a probability of less than one molecule, comparing them to multiple tiny reaction vessels, and binarizing each reaction vessel (signal of presence / absence of accompanying detection reaction) to count whether the target substance has entered the reaction vessel. Digital measurement can detect even one molecule of biomolecule, enabling high-precision quantitative determination. For example, Non-Patent Literature 1 discloses a method for detecting target nucleic acids that combines ICA (Invasive Cleavage Assay) with digital measurement.

[0004] Existing technical documents

[0005] Non-patent literature

[0006] Non-patent document 1: Journal of the Japan Society for Printing, 2017, Vol. 54, No. 6, pp. 377-382 Summary of the Invention

[0007] The problem that the invention aims to solve

[0008] Digital measurements are based on binarizing each reaction vessel (with / without the signal from the accompanying detection reaction). Therefore, to achieve higher precision in quantitative determination, a higher signal-to-noise ratio (S / N ratio) is required. Thus, the object of this invention is to provide a method for detecting target nucleic acids with an improved S / N ratio.

[0009] Methods for solving problems

[0010] This invention relates to a method for detecting a target nucleic acid, comprising: cleaving a first flap of a first split structure formed by a target nucleic acid, a first nucleic acid, and a second nucleic acid; cleaving a second flap of a second split structure formed by a third nucleic acid, the cleaved first flap, and a fourth nucleic acid; and detecting the presence of the target nucleic acid by detecting the cleaved second flap; wherein the steps of cleaving the first flap and the second flap are performed by cleaving the first flap and the second flap with a flanking endonuclease, the flanking endonuclease having an amino acid sequence having more than 96% sequence homology with the amino acid sequences of flanking endonucleases of bacteria selected from the group consisting of Thermococcus kodakarensis KOD1 strain, Pyrococcus abyssi GE5 strain, and Metha nothermobacter Thermautotrophicus Delta H strain.

[0011] In the detection method of the present invention, the first flank cleavage and the second flank cleavage are performed by a specific flank endonuclease, thus improving the S / N ratio compared with existing methods.

[0012] In the above detection methods, the target nucleic acid can be DNA.

[0013] In the above detection method, it is preferable to perform the steps of cutting off the first side wing and the second side wing under conditions where the pH is 7.5 or higher and 9.0 or lower. When the pH is within this range, the effect of improving the S / N ratio becomes more significant.

[0014] In the above detection method, it is preferable to perform the steps of cutting off the first side wing and the second side wing at a temperature of 55°C or higher and 70°C or lower. When the temperature is within this range, the effect of improving the S / N ratio becomes more significant.

[0015] In the above detection method, it is preferable to perform the steps of cutting off the first side wing and the second side wing in an aqueous solvent containing Mg. 2+ Additionally, Mg in aqueous solvents 2+ The concentration is more preferably 2.5 mM or higher and 20 mM or lower. As a result, the effect of improving the S / N ratio becomes more significant.

[0016] In the above detection method, it is preferable to perform the steps of cutting off the first side wing and cutting off the second side wing in an aqueous solvent, wherein the aqueous solvent contains K + The concentration is above 0 mM and below 100 mM. Therefore, the effect of improving the S / N ratio becomes more significant.

[0017] In the above detection method, it is preferable to perform the steps of cutting off the first side wing and the second side wing in an aqueous solvent, wherein the concentration of tris(hydroxymethyl)aminomethane in the aqueous solvent is greater than 0 mM and less than 300 mM. This makes the improvement in the S / N ratio more significant.

[0018] In the above detection method, the steps of cutting the first flanking segment and cutting the second flanking segment can be performed in an aqueous solvent, wherein the concentration of the flanking endonuclease in the aqueous solvent is 0.5 μM or higher and 8 μM or lower. This achieves a significant improvement in the S / N ratio.

[0019] In the above detection method, it is preferable to perform the steps of cutting off the first side wing and the second side wing in an aqueous solvent, wherein the volume of the aqueous solvent is 10 aL or more and 10 μL or less. This significantly improves the S / N ratio.

[0020] In the above detection method, the third nucleic acid and the fourth nucleic acid can be bound together by a linker molecule.

[0021] The above detection method can detect the severed second flank by detecting fluorescence.

[0022] In addition, the present invention also relates to a kit for use in the detection method of the present invention described above, comprising: the third nucleic acid, the fourth nucleic acid, the flanking endonuclease, and a specification showing instructions for designing the base sequences of the first and second nucleic acids based on the base sequence of the target nucleic acid.

[0023] The present invention also relates to an expression vector having a nucleic acid sequence encoding the following amino acid sequence and one or more regulatory sequences functionally linked to the above nucleic acid sequence, wherein the amino acid sequence has more than 96% sequence homology with the amino acid sequences of flanking endonucleases of bacteria selected from the group consisting of Thermococcus kodakarensis KOD1 strain, Pyrococcus abyssi GE5 strain, and Methanothermob acter Thermautotrophicus Delta H strain.

[0024] The expression vector described above can be used to prepare flanking endonucleases suitable for the detection method of the present invention. Alternatively, the expression vector can be a plasmid.

[0025] Invention Effects

[0026] According to the present invention, a method for detecting target nucleic acids with an improved S / N ratio can be provided. Attached Figure Description

[0027] Figure 1 This is an illustration of a cracked structure.

[0028] Figure 2 This is an explanatory diagram showing the summary of the evaluation system for Experimental Example 3.

[0029] Figure 3 This is a graph representing an example of the results of real-time measurement of fluorescence intensity in Experiment Example 3.

[0030] Figure 4 This is a graph representing the results of real-time fluorescence intensity measurements in Experiment Example 4.

[0031] Figure 5 This is a graph representing the results of real-time fluorescence intensity measurements in Experiment Example 4.

[0032] Figure 6 This is a graph representing the results of real-time fluorescence intensity measurements in Experiment Example 4.

[0033] Figure 7 This is a graph representing the results of real-time fluorescence intensity measurements in Experiment Example 4.

[0034] Figure 8 This is a graph representing the results of real-time fluorescence intensity measurements in Experiment Example 4.

[0035] Figure 9 This is a graph representing the results of real-time fluorescence intensity measurements in Experiment Example 4.

[0036] Figure 10 This is a graph representing the results of real-time fluorescence intensity measurements in Experiment Example 4.

[0037] Figure 11 This is a graph representing the results of real-time fluorescence intensity measurements in Experiment Example 4.

[0038] Figure 12 This is a graph representing the results of real-time fluorescence intensity measurements in Experiment Example 4.

[0039] Figure 13 This is a graph representing the results of real-time fluorescence intensity measurements in Experiment Example 4.

[0040] Figure 14 This is a photograph showing the results of the digital measurements in Experiment Example 5. Detailed Implementation

[0041] The following describes in detail the methods for implementing the present invention. However, the present invention is not limited to the following embodiments.

[0042] The target nucleic acid detection method of this embodiment includes: a step of cleaving a first flap of a first split structure formed by the target nucleic acid, a first nucleic acid, and a second nucleic acid (first flap cleavage step); a step of cleaving a second flap of a second split structure formed by a third nucleic acid, the cleaved first flap, and a fourth nucleic acid (second flap cleavage step); and a step of detecting the presence of the target nucleic acid by detecting the cleaved second flap (detection step). The cleavage of the first flap and the second flap is performed using specific flap endonucleases.

[0043] In this specification, a "cracked structure" refers to a structure consisting of a target nucleic acid and two nucleic acids capable of hybridizing with a first region and a second region adjacent to the target nucleic acid, respectively. A portion of the 3' side sequence of one nucleic acid (e.g., the first nucleic acid) hybridizes with the first region of the target nucleic acid, and the other nucleic acid (e.g., the second nucleic acid) hybridizes with the second region adjacent to the 3' side of the first region of the target nucleic acid. The remaining sequence on the 5' side of the nucleic acid hybridizing with the first region (e.g., the first nucleic acid) forms flanking fins with single-stranded protrusions. Specific examples of cracked structures include: Figure 1 The structure shown.

[0044] The flanking endonuclease (hereinafter also referred to as "FEN" or "FEN protein") used in the detection method of this embodiment only needs to have an amino acid sequence that has more than 96% sequence homology with the amino acid sequence of the flanking endonuclease of bacteria selected from the group consisting of Thermococcus kodakarensis KOD1 strain, Pyrococcus abyssi GE5 strain, and Methanothermobacter Thermautotrophicus Delta H strain. By using such a FEN, an improved S / N ratio can be achieved compared to existing methods.

[0045] The FEN used in the detection method of this embodiment only needs to have the activity of recognizing cracked structures, severing the root of the flanking wing (the phosphodiester bond on the 3' side of the overlapping site of three nucleic acids), and freeing the flanking wing (nucleic acid).

[0046] Among them, thermophilic archaea KOD1 strain, deep-sea thermococcus GE5 strain, and thermoautotrophic methanophilic thermobacter Delta H strain can be obtained from the RIKEN Center for Biological Resources Research (RIKEN BRC).

[0047] The FEN used in the detection method of this embodiment only needs to have an amino acid sequence homology of more than 96% with the amino acid sequence of the FEN of the above-mentioned strain. In addition, this FEN has the activity of recognizing the split structure, cleaving the root of the flanking part (the phosphodiester bond on the 3' side of the three overlapping nucleic acid sites), and freeing the flanking (nucleic acid).

[0048] In this specification, "sequence homology" refers to the percentage (%) of identical amino acid residues relative to the total overlapping amino acid sequence in the best alignment of two amino acid sequences using well-known mathematical algorithms in this art (preferably an algorithm that can consider introducing gaps into one or both sequences for optimal alignment). For example, NCBI BLAST (National Center for Biotechnology Information Basic Local Alignment Search Tool) can be used to prepare the alignment.

[0049] The amino acid sequence homology between the FEN used in the detection method of this embodiment and the amino acid sequence of the FEN possessed by the above-mentioned strain can be 96% or more, 97% or more, 98% or more, 99% or more, 99.1% or more, 99.2% or more, 99.3% or more, 99.4% or more, 99.5% or more, 99.6% or more, 99.7% or more, 99.8% or more, 99.9% or more, or even 100%.

[0050] The FEN protein used in the detection method of this embodiment can be obtained by extraction or purification and isolation from the above-mentioned strains, or by cloning the gene encoding the FEN protein from the above-mentioned strains, extracting or purifying the protein expressed in a heterologous protein expression system (recombinant protein expression system), and then isolating it. Alternatively, the above-mentioned strains can be omitted, and a gene encoding the amino acid sequence of the FEN protein possessed by the above-mentioned strains can be manufactured through chemical synthesis or the like, and the protein expressed in a heterologous protein expression system (recombinant protein expression system) can be extracted or purified and then isolated. When expressing the FEN protein in a heterologous protein expression system, a gene obtained by mutating the amino acid sequence in a manner that achieves the above-mentioned sequence homology range can also be used. Information such as the amino acid sequence of the FEN protein possessed by the above-mentioned strains can be obtained from sequence databases (e.g., GenBank, NCBI).

[0051] The first flanking severing step is the step of severing the first flanking of the first cleavage structure formed by the target nucleic acid, the first nucleic acid, and the second nucleic acid.

[0052] The first and second nucleic acids are designed to hybridize with the first region of the target nucleic acid and the second region adjacent to the 3' side of the first region, respectively. In this configuration, the first nucleic acid is designed so that a portion of the sequence on the 3' side hybridizes with the first region of the target nucleic acid, while the remaining sequence on the 5' side forms a single-stranded protruding flank. Furthermore, the flank sequence is designed to hybridize with the second region of the fourth nucleic acid. The second nucleic acid is designed to hybridize with the second region of the target nucleic acid. The first and second regions of the target nucleic acid are designed to have base sequences capable of specifically detecting the target nucleic acid. For example, in applications involving SNP analysis, it is preferable that the SNP site is located at the binding region between the first and second regions, i.e., at the overlapping site of the three nucleic acids.

[0053] The first split structure is as follows: a portion of the sequence on the 3' side of the first nucleic acid hybridizes with the first region of the target nucleic acid; the second nucleic acid hybridizes with the second region adjacent to the 3' side of the first region of the target nucleic acid; and the remaining sequence on the 5' side of the first nucleic acid protrudes as a single strand, forming a first flank. In the first flank cleavage step, FEN recognizes this first split structure and cleaves the root of the first flank (the phosphodiester bond on the 3' side of the overlapping site of the three nucleic acids), thus freeing the first flank (nucleic acid).

[0054] The first flank cutting step is preferably performed when the pH of the reaction solution is 7.5 or higher and 9.0 or lower. Within this pH range, the improvement in the S / N ratio becomes more significant. In addition to a good S / N ratio, from the viewpoint of enabling detection in a shorter time, the pH of the reaction solution is preferably in the range of 8.0 or higher and 9.0 or lower, more preferably in the range of 8.2 or higher and 8.8 or lower, and even more preferably in the range of 8.4 or higher and 8.6 or lower.

[0055] Preferably, the first flanking cut-off step is performed at a reaction solution temperature of 55°C or higher and 75°C or lower, more preferably at 55°C or higher and 70°C or lower. When the temperature is within this range, the improvement in the S / N ratio becomes more significant. In addition to a good S / N ratio, from the viewpoint of enabling detection in a shorter time even at low target nucleic acid concentrations, the reaction solution temperature can be, for example, 60°C or higher or 65°C or lower. When the reaction solution temperature is 60°C or higher and 65°C or lower, the effect of enabling detection in a shorter time even at low target nucleic acid concentrations is more significantly achieved.

[0056] The preferred first flank cutting step is performed in a process containing Mg 2+The reaction was carried out in an aqueous medium. From the perspective that the improvement in the S / N ratio becomes more significant, the Mg in the reaction solution (aqueous medium)... 2+ The concentration is preferably in the range of 1 mM or higher and 20 mM or lower, more preferably in the range of 2.5 mM or higher and 20 mM or lower. Besides a good S / N ratio, from the viewpoint of enabling detection in a shorter time, the Mg in the reaction solution (aqueous medium) is... 2+ The concentration is further preferably in the range of 5 mM or higher and 10 mM or lower. The Mg in the reaction solution (aqueous medium) 2+ The concentration can be adjusted, for example, by adding magnesium salts such as magnesium chloride to the reaction solution.

[0057] The preferred first flank cutting step is at K + The reaction was carried out in an aqueous medium with a concentration of 0 mM or higher but less than 100 mM. This resulted in a more significant improvement in the S / N ratio. It should be noted that within this range, even if the K concentration in the reaction solution (aqueous medium) is within a certain range... + At a concentration of 0 mM, the S / N ratio and the rate of increase did not differ significantly, therefore, the addition of potassium (K) is not necessarily required. + From the perspective of simplifying the reaction solution, K is preferred. + The concentration is 0 mM. The K in the reaction solution (aqueous medium) is... + The concentration can be adjusted, for example, by adding potassium salts such as potassium chloride to the reaction solution.

[0058] The first flanking cutoff step can be performed in any buffer solution. Examples of buffer solutions include Tris buffer, Bis-Tris buffer, and MOPS buffer. From the viewpoint that the improvement in the S / N ratio becomes more significant, the first flanking cutoff step is preferably performed in an aqueous solvent with a tris(hydroxymethyl)aminomethane concentration of more than 0 mM and less than 300 mM. In addition to the more significant improvement in the S / N ratio, from the viewpoint that detection can be performed in a shorter time, a tris(hydroxymethyl)aminomethane concentration in the reaction solution (aqueous medium) of more than 5 mM and less than 300 mM is more preferable.

[0059] In the first flanking cleavage step, the concentration of the flanking endonuclease in the reaction solution (aqueous medium) can be above 0.5 μM and below 8 μM. This allows for a significant improvement in the S / N ratio.

[0060] The first flank cutting step is preferably carried out with a reaction solution (aqueous solvent) volume of 10 aL or more but less than 10 μL. This makes the improvement in the S / N ratio more significant.

[0061] The concentrations of the first and second nucleic acids in the reaction solution can be appropriately set, for example, within the range of 1 nM or more and 10 μM or less.

[0062] In the first flanking severing step, the first flanking severing fold forms a second split structure with the third and fourth nucleic acids.

[0063] The second flank severing step is the step of severing the second flank of the second split structure formed by the third nucleic acid, the severed first flank, and the fourth nucleic acid.

[0064] The third nucleic acid is designed to hybridize with the first region of the fourth nucleic acid. Specifically, the third nucleic acid is designed so that a portion of its 3' side sequence hybridizes with the first region of the fourth nucleic acid, while the remaining 5' side sequence protrudes as a single strand to form a flanking region. Additionally, the first flanking region is designed to hybridize with the second region of the fourth nucleic acid adjacent to the 3' side of the first region.

[0065] The second split structure is as follows: a portion of the sequence on the 3' side of the third nucleic acid hybridizes with the first region of the fourth nucleic acid; the first flank hybridizes with the second region of the fourth nucleic acid adjacent to the 3' side of the first region; and the remaining sequence on the 5' side of the third nucleic acid protrudes as a single strand to form the second flank. During the second flank cleavage step, FEN recognizes this second split structure and cleaves the root of the second flank (the phosphodiester bond on the 3' side of the overlapping site of the three nucleic acids), thus freeing the second flank (nucleic acid).

[0066] The third and fourth nucleic acids can be formed as different nucleic acid molecules, or they can be linked together by linker molecules. Examples of linker molecules include nucleic acids (i.e., nucleic acids that are linked together by a linker molecule that is a nucleic acid, thus forming a single nucleic acid molecule) and spacers composed of chain-like molecules.

[0067] The third nucleic acid can be labeled according to the detection method in the detection steps. Examples of labels include fluorescent dyes, radionuclides, luminescent substances, phosphorescent substances, and redox molecules. The labeling method can follow conventional methods in this technical field. When using fluorescent dyes for labeling, for example, the fluorescent dye can be bound to a site corresponding to the flanking portion of the third nucleic acid, and an extinction molecule corresponding to the type of fluorescent dye can be bound to a portion of the sequence corresponding to the 3' side of the third nucleic acid. Thus, in the second cleaved structure state, since fluorescence from the fluorescent dye is not detected by FRET, fluorescence from the fluorescent dye is detected when the second flanking portion is free, thus enabling simpler detection.

[0068] Preferred reaction conditions in the second flank cutting step can be the same as those described in the first flank cutting step.

[0069] The concentrations of the third and fourth nucleic acids in the reaction solution can be appropriately set, for example, within the range of 1 nM or more and 10 μM or less.

[0070] The first and second flanking cleavage steps can be performed in different reaction vessels or in the same reaction vessel. For ease of operation, it is preferable to perform the first and second flanking cleavage steps in the same reaction vessel. In this case, the reaction solution can be pre-contained with the third and fourth nucleic acids.

[0071] The detection step is a step of detecting the presence of target nucleic acid by detecting the cleaved second flank. The detection step can be implemented, for example, by detecting bands of a size corresponding to the second flank via electrophoresis; or, if the second flank is labeled, by detecting based on that label using fluorescence or radioactivity.

[0072] The target nucleic acid detection method of this embodiment is applicable to digital measurement because of the improved S / N ratio.

[0073] The kit of this embodiment is for use in the detection method of the present invention described above, and comprises: the third nucleic acid, the fourth nucleic acid, the flanking endonuclease, and an instruction manual showing guidance on designing the base sequences of the first and second nucleic acids based on the base sequence of the target nucleic acid. The specific configurations of the first, second, third, and fourth nucleic acids are as described above.

[0074] In addition, the present invention relates to an expression vector having a nucleic acid sequence encoding the following amino acid sequence and one or more regulatory sequences functionally linked to the above nucleic acid sequence, wherein the amino acid sequence has more than 96% sequence homology with the amino acid sequences of flanking endonucleases of bacteria selected from the group consisting of Thermococcus kodakarensis KOD1 strain, Pyrococcus abyssi GE5 strain, and Methanot hermobacter Thermautotrophicus Delta H strain.

[0075] According to the expression vector of this embodiment, FEN protein suitable for the detection method of the present invention described above can be produced.

[0076] Regulatory sequences are sequences that control the expression of FEN proteins in the host (e.g., promoters, enhancers, ribosome-binding sequences, transcription termination sequences, etc.), and can be appropriately selected according to the type of host. The type of expression vector can be appropriately selected according to the type of host, such as plasmid vectors, viral vectors, granular vectors, Fors plasmid vectors, artificial chromosome vectors, etc.

[0077] Example

[0078] The present invention will now be described in more detail based on experimental examples. However, the present invention is not limited to the following experimental examples.

[0079] [Experimental Example 1: Preparation of FEN Protein]

[0080] Genes encoding the various flanking endonuclease (FEN) proteins shown in Table 1 were obtained by PCR or chemical synthesis, and the obtained genes were recombined into an expression vector (pET21a) for *E. coli*. In the case of genes obtained by chemical synthesis, the genes were synthesized by adding a 6-histidine residue to the C-terminus. The FEN protein in Experiment No. 1 (abbreviated as Afu) is the enzyme previously used in the ICA method (see Japanese Patent Publication No. 2001-518805).

[0081] Table 1

[0082]

[0083] The constructed expression vector was used to transform *Escherichia coli* (BL21-CodonPlus(DE3)). The transformed *E. coli* were inoculated into LB medium containing 100 μg / mL ampicillin and 35 μg / mL chloramphenicol, and cultured at 37°C until OD500. 600 The concentration was increased to approximately 0.5. Then, 1 M IPTG was added to achieve a final concentration of 0.5 mM, and the mixture was incubated at 25°C for 16 hours to induce high expression of the FEN protein.

[0084] Cultured *E. coli* (1 L of culture medium) was recovered by centrifugation and resuspended in buffer (25 mL). The resuspended *E. coli* was ultrasonically disrupted, and the supernatant was recovered by centrifugation. The recovered supernatant was then heated at 80°C for 20 minutes and centrifuged again. Polyethyleneimine was added to the recovered supernatant, and the supernatant was recovered by centrifugation. Ammonium sulfate was added to the recovered supernatant to achieve 80% saturation, and the resulting precipitate was recovered. The precipitate was resuspended in a buffer containing ammonium sulfate to remove insoluble matter, and fractionated using hydrophobic interaction chromatography to recover the fraction containing the target FEN protein. The recovered fraction was dialyzed against buffer, and the FEN protein was further purified by heparin affinity chromatography. For proteins with histidine residues at the C-terminus, cultured *E. coli* (1 L of culture medium) was recovered by centrifugation and resuspended in buffer (25 mL). The resuspended *E. coli* was ultrasonically disrupted, and the supernatant was recovered by centrifugation. Next, the recovered supernatant was heated at 60–80°C for 20 minutes and then centrifuged to recover the supernatant. The recovered supernatant was then added to TALON metal affinity resin (Clontech) and fractionated using immobilized metal affinity chromatography with an immobilized buffer containing imidazole to recover the fraction containing the target FEN protein.

[0085] It should be noted that the FEN proteins from Experiment No. 7 (Sso) and Experiment No. 8 (Sto) agglomerated and precipitated during the above purification process and could not be purified, so they were excluded from future studies.

[0086] The ultraviolet absorbance of each purified FEN protein was measured, and the concentration was calculated based on the molar absorptivity at a wavelength of 280 nm. Furthermore, SDS-PAGE analysis of each purified FEN protein confirmed the presence of a single band of the expected size. Although some small bands were detected, the absence of non-specific nuclease activity was confirmed.

[0087] [Experimental Example 2: Evaluation of Flanking Cut-off Activity]

[0088] The flanking cleavage activity of the purified FEN protein in Example 1 was evaluated. The cleavage structure was formed by the target nucleic acid (5'-GGTGATCGTTCGCTACATGTCGTCAGGATTCCAGGCAG-3': sequence number 1), the first nucleic acid (5'-FITC-AGACACATGGTATGTAGCGAACGATCACC-3': sequence number 2), and the second nucleic acid (5'-CTGCCTGGAATCCTGACGAC-3': sequence number 3). In this cleavage structure, a portion of the sequence on the 5' side of the first nucleic acid (11 bases from the 5' end to the underlined T) protruded as a single strand as a flanking fossa.

[0089] The cleavage reaction was carried out as follows: a reaction solution was prepared with 50 mM Tris-HCl (pH 7.6), 2.5 mM MgCl2, 10 nM substrate DNA (an equimolar mixture of the target nucleic acid, the first nucleic acid, and the second nucleic acid), and 0.5 μM FEN protein, and the reaction solution was incubated at 55 °C for 10 minutes.

[0090] After the reaction, electrophoresis was performed using a 12% polyacrylamide gel containing 8M urea. Next, the fluorescence intensity of the uncut cleaved structure (bands at 38 base positions) and the cleaved flanks (bands at 11 base positions) was measured using an image analyzer (Typhoon Trio+, GE Healthcare). The flank cleavage activity was calculated based on the measured fluorescence intensity using the following formula. The results are shown in Table 2.

[0091] Flanking cleavage activity = (fluorescence intensity of the band at the 11-base position) / {(fluorescence intensity of the band at the 11-base position) + (fluorescence intensity of the band at the 38-base position)} × 100 (%)

[0092] Table 2

[0093] 1 Afu 94 2 Ape 94 3 Mth 97 4 Pab 97 5 Tac 96 6 Tko 96 7 Sso - 8 Sto - 9 Pfuma 90 10 Pdela 93 11 Pisla 83 12 Pferr 84 13 Pogun 11 14 Shell 95 15 Clagu 54 16 Mjann 90 17 Mvulc 94 18 Migne 94 19 Gacet 82 20 Gahan 83 21 Aprof 89 22 Mwolf 93 23 Mmarb 91 24 Mthermo 97 25 Pfu 96 26 Phori 94 27 Tgorg 88 28 Tgamm 92 29 Tzill 84

[0094] As shown in Table 2, FEN proteins other than those tested in Experiment No. 13 (Pogun) and Experiment No. 15 (Clagu) were confirmed to have sufficient flanking cleavage activity.

[0095] [Experimental Example 3: Evaluation of Two-Stage Flanking Cut-Off Activity]

[0096] For the FEN protein whose flanking cleavage activity was confirmed in Experimental Example 2, the flanking cleavage activity in the two-stage flanking cleavage reaction typically used in the ICA assay was evaluated. A summary of the evaluation system is as follows: Figure 2 As shown. Figure 2The evaluation system shown is the same as that commonly used in the ICA method, except that it can detect the first-stage flanking cleavage reaction (the reaction that cleaves the first flanking of the first cleavage structure) based on the FEN protein by fluorescently labeling the allele probe.

[0097] exist Figure 2 In the evaluation system shown, the first cleavage structure is formed by the target nucleic acid (base sequence: 5'-GGTGATCGTTCGCTACATGTCGTCAGGATTCCAGGCAG-3': sequence number 4), the allele probe (base sequence: 5'-FAM-AGACACATGGTATGTAGCGAACGATCACC-BHQ1-3': sequence number 5) corresponding to the first and second nucleic acids, respectively, and the invading oligonucleotide (base sequence: 5'-CTGCCTGGAATCCTGACGAC-3': sequence number 6). Fluorescent molecules (FAM) and extinction molecules (BHQ1: a molecule modified with a black hole quencher on a thymine residue) are bound to the 5' and 3' ends of the allele probe, respectively. When the first flanking cleavage is performed by the FEN protein (the first stage of the flanking cleavage reaction), the green fluorescence of FAM can be detected. The cleaved first flanking fossa and the corresponding third and fourth nucleic acids (base sequence: 5'-RedmondRed-TCT-EclipseQuencher-TCGGCCTTTTGGCCGAGAGACTCCGCGTCCGT-3': SEQ ID NO. 7) formed a second cleavage structure. A fluorescent molecule (Redmond RED) is bound to the 5' end of the nucleic acid, and an extinction molecule (Eclipse Quencher) is inserted between the third and fourth residues from the 5' end of the nucleic acid. When the second flanking fossa is cleaved by the FEN protein (second-stage flanking cleavage reaction), the red fluorescence of Redmond RED can be detected.

[0098] For FEN proteins from tests No. 1 (Afu), No. 3 (Mth), No. 4 (Pab), No. 6 (Tko), and No. 25 (Pfu), a two-stage flanking cleavage reaction was evaluated. The two-stage flanking cleavage reaction was performed as follows: a reaction solution was prepared with the following composition: 1 μM allele probe (first nucleic acid), 1 μM invading oligonucleotide (second nucleic acid), 4 μM detection nucleic acid (third and fourth nucleic acids), 50 mM Tris-HCl (pH 8.5), 0.05 v / v%, Tween 20, 20 mM MgCl2, 0.086 mg / mL or 0.1 mg / mL FEN protein, and 0 M, 1.5 pM, 100 nM, or 1 μM target nucleic acid. Fluorescence intensity was measured in real-time using a LightCycler 480 (registered trademark) (manufactured by Roche Diagnostics), and the solution was incubated at 65°C for 60 minutes.

[0099] Figure 3 An example of the results of a real-time measurement of fluorescence intensity. Figure 3 These are the results obtained using FEN protein from Experiment No. 6 (Tko). Figure 3 (A) is a graph representing the measurement results of green fluorescence (fluorescence from the free first flanking edge). Figure 3 (B) is a graph showing the measurement results of red fluorescence (fluorescence from the free second flank).

[0100] like Figure 3 As shown, it can be confirmed that both green and red fluorescence tend to increase accordingly with the concentration of the target nucleic acid. Furthermore, at target nucleic acid concentrations of 100 nM and 1 μM, fluorescence intensity is considered saturated due to the high concentration of the target nucleic acid. Additionally, at a target nucleic acid concentration of 1.5 pM, compared to green fluorescence, the difference between red fluorescence and background (at a target nucleic acid concentration of 0 M) becomes larger. This is because the free first flanking segment repeatedly forms a second cleavage structure with the detection nucleic acid, thereby amplifying the signal (the free second flanking segment).

[0101] The S / N ratio was calculated by comparing the red fluorescence intensity (signal) at a target nucleic acid concentration of 1.5 pM to the red fluorescence intensity (noise) at a target nucleic acid concentration of 0 pM at a time point 30 minutes after the start of the reaction. The results are shown in Table 3.

[0102] Table 3

[0103] 1 Afu 3.2 3 Mth 3.3 4 Pab 4.8 6 Tko 5.3 25 Pfu 2.4

[0104] As shown in Table 3, the FEN proteins from tests No. 3 (Mth), No. 4 (Pab), and No. 6 (Tko) exhibited higher S / N ratios than the FEN protein from the previously used test No. 1 (Afu). On the other hand, the FEN protein from test No. 25 (Pfu) had a lower S / N ratio than the FEN protein from the previously used test No. 1 (Afu).

[0105] For FEN proteins other than those mentioned above and FEN proteins from Test No. 6 (Tko), the two-stage flanking cleavage reaction was evaluated under the following conditions. The two-stage flanking cleavage reaction was performed as follows: a reaction solution was prepared with the following composition: 1 μM allele probe (first nucleic acid), 1 μM invading oligonucleotide (second nucleic acid), 2 μM detection nucleic acid (third and fourth nucleic acids), 50 mM Tris-HCl (pH 8.5), 0.05 v / v% Tween 20, 2.5 mM MgCl2, 1.16 μM (Test No. 10 only) or 7.74 μM FEN protein, 0 M, 1.5 pM, 30 pM or 100 pM target nucleic acid. Fluorescence intensity was measured in real time using a LightCycler 480 (registered trademark) (manufactured by Roche Diagnostics), and the solution was incubated at 65°C for 60 minutes.

[0106] The S / N ratio was calculated by comparing the red fluorescence intensity (signal) at a target nucleic acid concentration of 1.5 pM to the red fluorescence intensity (noise) at a target nucleic acid concentration of 0 pM at a time point 30 minutes after the start of the reaction. The results are shown in Table 4.

[0107] Table 4

[0108]

[0109] FEN proteins in experiments No. 9–24 and No. 26–29 did not show a high S / N ratio.

[0110] [Experimental Example 4: Study on the reaction conditions of the flanking cutoff reaction]

[0111] Using FEN proteins from Experiment No. 3 (Mth) and Experiment No. 6 (Tko), the reaction conditions for the flanking cleavage reaction were studied in the same evaluation system as in Experiment No. 3.

[0112] <ph>

[0113] The reaction solution was prepared as follows: based on 50 mM Tris-HCl adjusted to pH 7.5, 8.0, 8.5, or 9.0, containing 1 μM allele probe (first nucleic acid), 1 μM intrusion oligonucleotide (second nucleic acid), 2 μM detection nucleic acids (third and fourth nucleic acids), 0.05 v / v% Tween 20, 2.5 mM MgCl2, 0.5 μM FEN protein, and 0 M, 100 pM, or 1 nM target nucleic acid. The reaction volume was 10 μL. After preparing the reaction solution, fluorescence intensity was measured in real time using a LightCycler 480 (registered trademark) (manufactured by Roche Diagnostics), while incubation at 67°C for 60 minutes to perform flanking cleavage. It should be noted that in the pH study, only FEN protein from assay No. 6 (Tko) was used.

[0114] The S / N ratio was calculated based on the ratio of the red fluorescence intensity (signal) at a target nucleic acid concentration of 100 pM to the red fluorescence intensity (noise) at a target nucleic acid concentration of 0 pM at a time point 30 minutes after the start of the reaction. The results are shown in Table 5. Additionally, Figure 4 The graph shows the real-time measurement results of fluorescence intensity. The vertical axis of the graph represents fluorescence intensity, and the horizontal axis represents the time (in minutes) from the start of the reaction. Figure 4 (A)~(D) represent the results obtained by measuring FEN protein using Test No. 6 (abbreviated as Tko) at pH 7.5, pH 8.0, pH 8.5 or pH 9.0.

[0115] Table 5

[0116]

[0117] like Figure 4 As shown in Table 5, a good S / N ratio is observed within the pH range of the reaction solution, above 7.5 and below 9.0. Furthermore, near pH 8.5, in addition to the good S / N ratio, a faster increase in red fluorescence intensity is also observed. The faster the increase in red fluorescence intensity, the shorter the detection time.

[0118] <Temperature>

[0119] The reaction solution consisted of: 1 μM allele probe (first nucleic acid), 1 μM invading oligonucleotide (second nucleic acid), 2 μM detection nucleic acid (third and fourth nucleic acids), 50 mM Tris-HCl (pH 8.5), 0.05 v / v% Tween 20, 2.5 mM MgCl2, 0.5 μM FEN protein, and 0 M, 30 pM, or 1 nM target nucleic acid. The reaction volume was 10 μL. After preparing the reaction solution, the fluorescence intensity was measured in real time using a LightCycler 480 (registered trademark) (manufactured by Roche Diagnostics), and flanking cleavage was performed by incubation at 55°C, 60°C, 65°C, 67°C, or 70°C for 60 minutes.

[0120] The S / N ratio was calculated by comparing the red fluorescence intensity (signal) at a target nucleic acid concentration of 30 pM or 1 nM at 30 minutes after the start of the reaction with the red fluorescence intensity (noise) at a target nucleic acid concentration of 0 M. The results are shown in Table 6. Additionally, Figures 5-8 The results of real-time fluorescence intensity measurement are shown in a graph. The vertical axis of the graph represents fluorescence intensity, and the horizontal axis represents the time (in minutes) from the start of the reaction. Figure 5 (A)~(E) represent the results obtained by measuring the FEN protein of test No. 6 (abbreviated as Tko) at a concentration of 30 pM of target nucleic acid at 55℃, 60℃, 65℃, 67℃ or 70℃. Figure 6 (A)~(E) represent the results obtained by measuring the FE N protein of test No. 6 (abbreviated as Tko) at a concentration of 1 nM of target nucleic acid at 55℃, 60℃, 65℃, 67℃ or 70℃. Figure 7 (A)~(E) represent the results obtained by measuring the FEN protein of test No.3 (abbreviated as Mth) at a concentration of 30pM of target nucleic acid at 55℃, 60℃, 65℃, 67℃ or 70℃. Figure 8 (A)~(E) represent the results obtained by measuring the FEN protein of test No.3 (abbreviated as Mth) at a concentration of 1 nM of target nucleic acid at 55℃, 60℃, 65℃, 67℃ or 70℃.

[0121] Table 6

[0122]

[0123] like Figures 5-8 As shown in Table 6, a good S / N ratio was observed within a reaction solution temperature range of 55°C to 70°C. Furthermore, in the FEN protein of Experiment No. 6 (Tko), in addition to a good S / N ratio, a faster increase in red fluorescence intensity was observed even at low target nucleic acid concentrations within a temperature range of 60°C to 70°C. The same effect was observed in the FEN protein of Experiment No. 3 (Mth) within a temperature range of 55°C to 65°C.

[0124] <Mg 2+ Ion concentration >

[0125] The reaction solution was prepared as follows: 1 μM allele probe (first nucleic acid), 1 μM intrusion oligonucleotide (second nucleic acid), 2 μM detection nucleic acid (third and fourth nucleic acids), 50 mM Tris-HCl (pH 8.5), 0.05 v / v% Tween 20, 1 mM, 2.5 mM, 5 mM, 10 mM or 20 mM MgCl2, 0.5 μM FEN protein, 0 M, 100 pM or 1 nM target nucleic acid. The reaction volume was 10 μL. After preparing the reaction solution, the fluorescence intensity was measured in real time using a LightCycler 480 (registered trademark) (manufactured by Roche Diagnostics), and the solution was incubated at 67 °C for 60 minutes to perform a flanking cleavage reaction. It should be noted that in Mg... 2+ In the study of ion concentration, only the FEN protein from Experiment No. 6 (Tko) was used.

[0126] The S / N ratio was calculated based on the ratio of the red fluorescence intensity (signal) at a target nucleic acid concentration of 100 pM to the red fluorescence intensity (noise) at a target nucleic acid concentration of 0 pM at a time point 30 minutes after the start of the reaction. The results are shown in Table 7. Additionally, Figure 9 The results of real-time fluorescence intensity measurement are shown in a graph. The vertical axis of the graph represents fluorescence intensity, and the horizontal axis represents the time (in minutes) from the start of the reaction. Figure 9 (A)~(E) indicate the use of FEN protein from Test No. 6 (abbreviated as Tko) at 1mM, 2.5mM, 5mM, 10mM or 20mM Mg 2+ The results of concentration measurement.

[0127] Table 7

[0128]

[0129] like Figure 9 And as shown in Table 7, Mg in the reaction solution 2+ A good S / N ratio is observed in concentrations of 1 mM or higher and 20 mM or lower (preferably 2.5 mM or higher and 20 mM or lower). Furthermore, in Mg... 2+ In the concentration range of 5 mM to 10 mM, in addition to a good S / N ratio, a faster increase in red fluorescence intensity is also observed.

[0130] <K + Ion concentration >

[0131] The reaction solution consisted of: 1 μM allele probe (first nucleic acid), 1 μM invading oligonucleotide (second nucleic acid), 2 μM detection nucleic acid (third and fourth nucleic acids), 50 mM Tris-HCl (pH 8.5), 0.05 v / v% Tween 20, 2.5 mM MgCl2, 0 mM, 25 mM, 50 mM, 100 mM or 200 mM KCl, 0.5 μM FEN protein, and 0 M, 1 nM or 10 nM target nucleic acid. The reaction volume was 10 μL. After preparing the reaction solution, the fluorescence intensity was measured in real time using a LightCycler 480 (registered trademark) (manufactured by Roche Diagnostics), and the solution was incubated at 67 °C for 60 minutes to perform a flanking cleavage reaction.

[0132] The S / N ratio was calculated based on the ratio of the red fluorescence intensity (signal) at a target nucleic acid concentration of 1 nM to the red fluorescence intensity (noise) at a target nucleic acid concentration of 0 M at a time point 30 minutes after the start of the reaction. The results are shown in Table 8. Additionally, Figures 10-11 The results of real-time fluorescence intensity measurement are shown in a graph. The vertical axis of the graph represents fluorescence intensity, and the horizontal axis represents the time (in minutes) from the start of the reaction. Figure 10 (A)~(E) indicate the use of FEN protein from test No. 6 (abbreviated as Tko) at 0mM, 25mM, 50mM, 100mM or 200mM K. + The results of concentration measurement. Figure 11 (A)~(E) indicate the use of FEN protein from test No. 3 (abbreviated as Mth) at 0mM, 25mM, 50mM, 100mM or 200mM K. + The results of concentration measurement.

[0133] Table 8

[0134]

[0135] like Figures 10-11 And as shown in Table 8, K in the reaction solution + A good S / N ratio is observed in the concentration range of 100 mM or less. In addition, within this range, there is no significant difference in the S / N ratio and the rising speed, so it is found that K + is not necessarily required (may be 0 mM).

[0136] <Tris concentration>

[0137] The reaction solution was composed of: 1 μM allele probe (first nucleic acid), 1 μM invasive oligonucleotide (second nucleic acid), 2 μM detection nucleic acid (third nucleic acid and fourth nucleic acid), 5 mM, 20 mM, 50 mM, 100 mM, 200 mM, 300 mM, 400 mM or 500 mM Tris-HCl (pH 8.5), 0.05 v / v% Tween 20, 5 mM MgCl₂, 0.5 μM FEN protein, 0 M, 100 pM or 1 nM target nucleic acid. The volume of the reaction solution was set to 10 μL. After preparing the reaction solution, the fluorescence intensity was measured in real time using LightCycler 480 (registered trademark) (manufactured by Roche Diagnostics), and incubation was performed at 67°C for 60 minutes to carry out the flap cleavage reaction.

[0138] The S / N ratio was calculated as the ratio of the red fluorescence intensity (signal) when the target nucleic acid concentration was 100 pM to the red fluorescence intensity (noise) when the target nucleic acid concentration was 0 M, at the time point 30 minutes after the start of the reaction. The results are shown in Table 9. In addition, Figures 12-13 shows the results (graphs) of real-time measurement of fluorescence intensity. The vertical axis of the graph represents the fluorescence intensity, and the horizontal axis represents the time (minutes) elapsed from the start of the reaction, Figure 12 (A) to (H) represent the measurement results obtained using the FEN protein of Test No. 6 (abbreviated as Tko) at Tris concentrations of 5 mM, 20 mM, 50 mM, 100 mM, 200 mM, 300 mM, 400 mM, or 500 mM. Figure 13 (A) to (H) represent the measurement results obtained using the FEN protein of Test No. 3 (abbreviated as Mth) at Tris concentrations of 5 mM, 20 mM, 50 mM, 100 mM, 200 mM, 300 mM, 400 mM, or 500 mM.

[0139] Table 9

[0140]

[0141] As shown in Figures 12-13 and Table 9, a good S / N ratio is observed in the range where the Tris concentration in the reaction solution is 300 mM or less (preferably in the range of 5 mM to 300 mM). In addition, the rise of red fluorescence intensity is also faster.

[0142] [Experimental Example 5: Application in Digital Measurement]

[0143] The detection efficiency of target nucleic acids based on digital measurements was evaluated using FEN proteins from Experiment No. 1 (Afu) and Experiment No. 6 (Tko).

[0144] A microfluidic device is fabricated by bonding a plate-shaped, transparent cyclic olefin polymer (COP) substrate with multiple micropores to a COP-based cap component. The cap component is a component with an inlet and an outlet, designed to clamp the area where micropores are formed during bonding, and features a 50 μm raised step at its periphery. Each pore in the microfluidic device is formed into a cylindrical shape. The diameter (opening diameter) of each pore is designed to be 10 μm, and the depth is designed to be 15 μm. The substrate and cap component are fabricated using thermoplastic resin via injection molding. The ends of the step are brought into contact with the substrate, and the contact area is laser-fused together.

[0145] As reagent 1, a mixture containing 150 aM target DNA oligomer (Fasmac: target nucleic acid: 5'-CCGAAGGGCATGAGCTGCATGATGAGCTGCACGGTGGAGGTGAGGCAGATGCCCAG-3': SEQ ID NO. 8), 0.5 μM allele probe (Fasmac: first nucleic acid: 5'-ACGGACGCGGAGTGCAGCTCATGCCC-3': SEQ ID NO. 9), 1 μM intrusion oligomer (Fasmac: second nucleic acid: 5'-CCACCGTGCARCTCATCAA-3': SEQ ID NO. 10), 4 μM FRETCassette (Redmond RED-Eclipse Quanrure) (Tsukuba Oligo Service: third and fourth nucleic acids: 5'-Redmond Red-TCT-Eclipse Quencher-TCGGCCTTTTGGCCGAGAGACTCCGCGTCCGT-3': SEQ ID NO. 11), FEN protein (abbreviated as Tko or Afu), and 50 mM An aqueous solution of Tris-HCl (pH 8.5), 20 mM MgCl2, and 0.05 v / v% Tween 20 was used. Additionally, a fluorinated oil (FC40, manufactured by SIGMA) was used as the sealing fluid.

[0146] First, using a pipette, dispense 20 μL of the prepared reagent 1 into the inlet of the microfluidic device, filling the micropores and flow path with reagent 1. Then, dispense 150 μL of fluorinated oil into the inlet of the microfluidic device, replacing reagent 1 in the flow path and sealing reagent 1 within the micropores. The remaining reagent 1 and fluorinated oil are discharged from the outlet.

[0147] The microfluidic device was placed on a hot plate, and the plate was heated to 67°C for 25 minutes. The microfluidic device was then observed using a fluorescence microscope (BZ-X700, KEYENCE). The results are presented below. Figure 14 .

[0148] Based on preliminary experiments, the predicted number of bright spots, determined by the amount of target nucleic acid (150 aM), is (1 well / 13513 wells). Figure 14 As shown, the results for FEN protein in Experiment No. 6 (Tko) were largely consistent with the expected values. On the other hand, the results for FEN protein in Experiment No. 1 (Afu) significantly exceeded the predicted values, with most of the results being blurry brightness (side effects). < / ph>

Claims

1. A method for detecting target nucleic acids for non-disease diagnostic purposes, comprising: The step of cutting off the first flank of the first split structure formed by the target nucleic acid, the first nucleic acid, and the second nucleic acid; The step of cutting off the second wing of the second split structure formed by the third nucleic acid, the cut first wing, and the fourth nucleic acid; as well as The step of detecting the presence of target nucleic acid by detecting the severed second flank; A portion of the sequence on the 3' side of the first nucleic acid hybridizes with the first region of the target nucleic acid, and the second nucleic acid hybridizes with the second region of the target nucleic acid adjacent to the 3' side of the first region. The remaining sequence on the 5' side of the first nucleic acid protrudes as a single strand, forming the first flanking region. A portion of the sequence on the 3' side of the third nucleic acid hybridizes with the first region of the fourth nucleic acid, the first flank hybridizes with the second region adjacent to the 3' side of the first region of the fourth nucleic acid, and the remaining sequence on the 5' side of the third nucleic acid protrudes as a single strand to form the second flank; The steps of cutting off the first flank and the second flank are performed by cutting off the first flank and the second flank using a flank endonuclease. The steps of cutting off the first flank and the steps of cutting off the second flank are performed under conditions of pH 7.5 or higher and 9.0 or lower, and temperature 55°C or higher and 70°C or lower, in Mg 2+ The reaction was carried out in an aqueous solvent with a concentration of 2.5 mM or higher but less than 20 mM. The flanking endonuclease is the flanking endonuclease of the thermophilic archaea (Thermococcus kodakarensis) strain KOD1, with accession number TK_RS06335. The target nucleic acid is DNA.

2. The method according to claim 1, wherein, K in the aqueous solvent + The concentration is above 0 mM and below 100 mM.

3. The method according to claim 1 or 2, wherein, The concentration of trihydroxymethylaminomethane in the aqueous solvent is greater than 0 mM and less than 300 mM.

4. The method according to claim 1 or 2, wherein, The concentration of the flanking endonuclease in the aqueous solvent is above 0.5 μM and below 8 μM.

5. The method according to claim 1 or 2, wherein, The volume of the aqueous solvent is 10aL or more and 10μL or less.

6. The method according to claim 1 or 2, wherein, The third nucleic acid and the fourth nucleic acid are linked by a linker molecule.

7. The method according to claim 1 or 2, wherein, The severed second flank was detected by detecting fluorescence.

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