Method for detecting target substance using aptamer beacon

Abstract

The present application aims to provide a means for enhancing the sensitivity of a nucleic acid aptamer beacon. In one aspect, the present application provides the following technical solutions. A method for detecting a target substance using a nucleic acid aptamer beacon, wherein the method comprises: (A) a step of mixing the target substance, the nucleic acid aptamer beacon, and a response-enhancing nucleic acid; and (B) a step of measuring luminescence from the mixture, the response-enhancing nucleic acid comprising a region capable of complementary binding to a portion of at least either of a region of a first nucleic acid and a region of a second nucleic acid of the nucleic acid aptamer beacon.

Description

Technical Field

[0001] This invention relates to a method for detecting target substances using nucleic acid aptamer beacons. Background Technology

[0002] To achieve the goal of detecting target substances, various analytical methods are utilized. One such method involves using nucleic acid aptamers. Nucleic acid aptamers possess nucleic acid sequences capable of specifically binding to target substances. Furthermore, binding to the target substance induces a structural change. Nucleic acid aptamer beacons utilize this structural change. Specifically, nucleic acid aptamer beacons rely on structural changes to alter the degree of luminescence.

[0003] Patent Document 1 discloses a mechanism for causing changes in the degree of luminescence. Specifically, Patent Document 1 discloses a first reporter group and a second reporter group, wherein the first reporter group is a phosphor and the second reporter group is a chemically extinct group. When a structural change is caused, the distance between the two reporter groups changes, thereby causing a change in the degree of luminescence.

[0004] [Existing technical documents] [Patent Literature]

[0005] Patent Document 1: International Publication No. 00 / 070329 (Japanese Invention Patent Publication No. 2003-508729) Summary of the Invention

[0006] [The technical problem that the invention aims to solve]

[0007] Nucleic acid aptamer beacons are typically manufactured using the following steps.

[0008] • Screening of nucleic acids based on the interaction between the target substance and nucleic acid.

[0009] • Candidate nucleic acids are isolated based on the screening results.

[0010] • Since the isolated candidate nucleic acid can function as a nucleic acid aptamer or has a high probability of functioning as a nucleic acid aptamer, a nucleic acid aptamer beacon is made from this nucleic acid.

[0011] However, in practice, in some cases, it is sometimes impossible to obtain nucleic acid aptamers that demonstrate the performance required to achieve the intended purpose. For example, in some cases, although the nucleic acid aptamer binds to the target substance, its sensitivity as a nucleic acid aptamer beacon is sometimes insufficient.

[0012] In such a situation, searching for candidate nucleic acids with different sequences would require a significant investment of effort.

[0013] Based on the above reasons, the object of the present invention is to provide a means to enhance the sensitivity of nucleic acid aptamer beacons.

[0014] [Technical solutions used to solve technical problems]

[0015] To achieve the above objectives, the present invention includes the following invention in one embodiment.

[0016] (Invention 1) A method for detecting target substances using nucleic acid aptamer beacons, wherein, The nucleic acid aptamer beacon has the following characteristics: (1) The region of the first substance emits fluorescence; (2) The region of the second substance, which modulates the luminescence produced by the first substance; (3) The region of the first nucleic acid, which is directly or indirectly connected to the first substance; (4) The region of the second nucleic acid, which is directly or indirectly linked to the second substance; and (5) The target substance binding region, which binds to the target substance and can cause structural changes in the nucleic acid aptamer beacon. The regions of the first nucleic acid and the second nucleic acid can be integrated, or they can exist separately. The regions of the first nucleic acid and the second nucleic acid have at least a portion of complementary sequences that can bind to each other. The method includes: (A) The step of mixing the target substance, the nucleic acid aptamer beacon, and the nucleic acid for response enhancement; and (B) The procedure for measuring the luminescence from the mixture. The nucleic acid for enhancing the response includes a region capable of complementary binding to a portion of a region of at least one of the regions of the first nucleic acid and the second nucleic acid.

[0017] (Invention 2) According to the method of invention 1, the first substance and the second substance are a combination of donor fluorescence extinguished by FRET.

[0018] (Invention 3) According to the method of invention 1, the first substance and the second substance are a combination of fluorescent expression generated by FRET-induced receptor.

[0019] (Invention 4) The method according to any one of inventions 1 to 3, wherein the nucleic acid is DNA.

[0020] (Invention 5) The method according to any one of inventions 1 to 3, wherein the nucleic acid is RNA.

[0021] (Invention 6) According to any one of the inventions 1 to 5, the nucleic acid used for response enhancement has a base length of 8 bases or more.

[0022] [Invention Effects]

[0023] In one embodiment, the method of the present invention uses nucleic acid for response enhancement. This results in increased luminescence intensity and enhanced sensitivity. Attached Figure Description

[0024] Figure 1 This describes the principle of the nucleic acid aptamer beacon in one implementation method.

[0025] Figure 2 This illustrates the principle of nucleic acid aptamer beacons and nucleic acid for response enhancement in one implementation method.

[0026] Figure 3 This indicates the effect of the base length of oligomeric DNA on the fluorescence response of DNA aptamer beacons.

[0027] Oligo DNA of varying lengths was added to an adenosine detection system to compare the response to adenosine from DNA aptamer beacons. Oligo DNA-1 (A), Oligo DNA-2 (B), and Oligo DNA-3 (C) were added at final concentrations of 0, 5, 20, or 80 nM. Data are presented as mean ± standard deviation (n=3).

[0028] Figure 4 The effect of oligomeric DNA concentration on the fluorescence intensity change of DNA aptamer beacons over time is shown. The changes in fluorescence intensity over time in adenosine detection systems with the addition of 0 nM (A), 20 nM (B), and 80 nM (C) Oligo DNA-1 are illustrated. Data represent averages (n=3).

[0029] Figure 5 Standard curves and Hanes-Woolf scatter plots for adenosine. (A) Standard curves for adenosine with 0, 20, or 80 nM OligoDNA-1. Data are presented as mean ± standard deviation (n=3). (B) Coefficient of determination (R²) on the Hanes-Woolf scatter plot above the lower limit of quantitation in the data used in (A). 2 The regression line was constructed using data points in the concentration range (5-50 μM) above 0.99.

[0030] [Explanation of reference numerals in the attached figures] 10: Region of the first substance; 20: Region of the second substance; 30: Region of the first nucleic acid; 40: Region of the second nucleic acid; 50: Target substance binding region; 60: Target substance; 70: Nucleic acid for response enhancement. Detailed Implementation

[0031] The following describes specific embodiments for carrying out the invention. This description is intended to facilitate understanding of the invention and is not intended to limit the scope of the invention.

[0032] 1. Summary In one embodiment, the present invention relates to a method for detecting a target substance using nucleic acid aptamer beacons. The method includes the following steps.

[0033] (A) The steps for mixing the target substance, nucleic acid aptamer beacon, and nucleic acid for response enhancement; and (B) The procedure for measuring the luminescence from the mixture.

[0034] Here, the nucleic acid for response enhancement includes a region capable of complementary binding to a portion of at least one of the regions of the first and second nucleic acids of the nucleic acid aptamer beacon.

[0035] The following details each step and the elements required to perform that step.

[0036] 2. Nucleic acid aptamer beacons In the above method, nucleic acid aptamer beacons are used. Functionally, nucleic acid aptamer beacons have the following functions: they induce structural changes in nucleic acids upon binding to target substances, and can provide a notification in a form that is easily identifiable by these changes.

[0037] In one embodiment, the nucleic acid aptamer beacon has the following components (see reference). Figure 1 ).

[0038] • Substance 1 (10) • Substance 2 (20) • Region of the first nucleic acid (30) • Region of the second nucleic acid (40) • Target material binding region (50).

[0039] Substance 1 (10) has the function of fluorescence. Here, fluorescence means absorbing light of a certain wavelength and emitting light of another wavelength. In addition, from the viewpoint of the phenomenon known as FRET (Fluorescence Resonance Energy Transfer), substance 1 (10) can act as a donor molecule.

[0040] The second substance (20) has the function of regulating the luminescence of the first substance (10). For example, the second substance (20) may function as a quencher (also referred to as an extinction agent in this specification). Specifically, the second substance (20) is able to absorb light of a specific wavelength emitted by the first substance (10), and may not emit light of that wavelength or other wavelengths. Thus, the luminescence of the first substance (10) can be extinguished by the second substance (20).

[0041] Furthermore, from the perspective of a phenomenon known as FRET (Fluorescence Resonance Energy Transfer), the second substance (20) can function as an acceptor molecule. Specifically, the second substance (20) can absorb light of a specific wavelength emitted by the first substance (10), and can also emit light of other wavelengths.

[0042] The first nucleic acid region (30) is a region of nucleic acid that is directly or indirectly linked to the first substance (10). Moreover, the second nucleic acid region (40) is a region of nucleic acid that is directly or indirectly linked to the second substance (20).

[0043] In one example, "directly linked" means, for example, that the first substance (10), which is a substance other than nucleic acid, is not linked to the region (30) of the first nucleic acid, which is nucleic acid, without any other atoms or molecules. The same applies to the second substance (20).

[0044] In other examples, “indirect linking” means, for example, that a first substance (10), which is a substance other than a nucleic acid, is linked to a region (30) of a first nucleic acid via other substances (e.g., other atoms, other molecules, such as other nucleic acids or other linking molecules). The same applies to the second substance (20).

[0045] The target substance binding region (50) is a nucleic acid region that binds to the target substance (60). Preferably, it is a nucleic acid region that specifically binds to the target substance (60). Although not limited, specific binding means, for example, the degree to which the properties of the binding to the target substance (60) (e.g., binding constant, etc.) differ significantly from those of a control. Furthermore, the target substance binding region (50) and the region (30) of the first nucleic acid may be composed of different nucleic acid sequences, or the sequences of the two may be repeated (in other words, a part of the target substance binding region (50) may constitute a part of the region (30) of the first nucleic acid).

[0046] Combination Figure 1 To illustrate how the above-mentioned elements contribute to the detection of the target substance (60).

[0047] In one example, the region (30) of the first nucleic acid and the region (40) of the second nucleic acid bind complementaryly. This shortens the distance between the first substance (10) and the second substance (20) which are respectively linked to these regions. Therefore, the luminescence from the first substance (10) is modulated by the second substance (20). For example, when the second substance (20) functions as a quencher, the luminescence from the first substance (10) is extinguished.

[0048] However, in the presence of the target substance (60), the target substance (60) binds to the target substance binding region (50). This causes at least a localized structural change in the nucleic acid aptamer beacon. Furthermore, this structural change hinders the complementary binding of the region (30) of the first nucleic acid and the region (40) of the second nucleic acid. This increases the distance between the first substance (10) and the second substance (20). Therefore, the luminescence from the first substance (10) is not modulated by the second substance (20).

[0049] The above is just one example, and there are other possibilities based on... Figure 1 The various modification patterns of the structure are shown.

[0050] 2-1. Types of nucleic acids (Change of pattern 1) The nucleic acid used can be DNA, RNA, or a combination of both (e.g., the region (30) of the first nucleic acid is DNA and the region (40) of the second nucleic acid is RNA or the region (30) of the first nucleic acid is RNA and the region (40) of the second nucleic acid is DNA).

[0051] 2-2. Functions of Substance 1 and Substance 2 (Changing Mode 2) In one example, the first substance (10) and the second substance (20) may be a combination that causes the donor fluorescence to be extinguished via FRET. Here, extinction also includes the case of localized fluorescence attenuation. For example, the first substance (10) has the function of emitting light of a certain wavelength. Moreover, the second substance (20) has the function of absorbing light of that wavelength or near that wavelength (and the second substance (20) does not have the function of emitting light).

[0052] In other examples, the first substance (10) and the second substance (20) may be a combination of receptor-induced fluorescence expression via FRET (Fluorescence Resonance Energy Transfer). Here, the first substance (10) functions as a donor molecule, and the second substance (20) functions as an acceptor molecule. For example, the first substance (10) has the function of emitting light of a certain wavelength. Moreover, the second substance (20) has the function of absorbing light of that wavelength or near that wavelength. In addition, the second substance (20) can utilize light of that wavelength or near that wavelength as excitation light and has the function of emitting light of other wavelengths.

[0053] As another example, the first substance (10) and the second substance (20) can also act as donors and acceptors in PET (Photoinduced Electron Transfer).

[0054] 2-3. Regions of the first and second nucleic acids (modification of pattern 3) The regions of the first nucleic acid (30) and the second nucleic acid (40) can be integrated or exist separately. Figure 1 This is an example of the latter. In the case of integration, for example, the region of the first nucleic acid (30) and the region of the second nucleic acid (40) can exist separated by the target substance binding region (50) and / or other nucleic acid regions. When the two are at least locally complementary, the nucleic acid aptamer beacon can have a ring structure.

[0055] 2-4. Distance between Substance 1 and Substance 2 (Change Mode 4) exist Figure 1 In one example, when the target substance (60) is absent, the distance between the first substance (10) and the second substance (20) is brought closer, and when the target substance (60) is present, the distance between the first substance (10) and the second substance (20) is increased. In other examples, when the target substance (60) is absent, the distance between the first substance (10) and the second substance (20) can be increased, and when the target substance (60) is present, the distance between the first substance (10) and the second substance (20) can be brought closer.

[0056] As mentioned above, nucleic acid aptamer beacons can be modified in various alteration patterns or combinations thereof.

[0057] 2-5. Combination of Substance 1 and Substance 2 The combination of the first and second substances is not particularly limited; for example, it can be a combination capable of inducing FRET. When considering homogeneous FRET, the combination of the first and second substances can include not only combinations of different kinds of substances but also combinations of the same kind of substances. However, typically, the combination of the first and second substances is a combination of different kinds of substances. Additionally, the second substance may contain a matting agent.

[0058] Although there is no limitation, records may include, for example, combinations of the following.

[0059] CFP and YFP GFP (including EGFP) and mCherry mTurquoise2 and mVenus mNeonGreen and mRuby3 Fluorescein and Tetramethylrhodamine Fluorescein and Black Hole Quencher (registered trademark) 1 (BHQ-1) IAEDANS (5-[2-iodoacetyl]aminonaphthalene-1-sulfonic acid) and fluorescein EDANS (5-(2-aminoethylamino)naphthalene-1-sulfonic acid) and Dabcyl Cy3 and Cy5 Alexa Fluor 488 and Alexa Fluor 594.

[0060] In other examples, it could be a combination that can induce PET. Although there is no limitation, records may include, for example, combinations of the following.

[0061] Porphyrins and quinones Organic pigments and amines.

[0062] 3. Target material There are no particular limitations on the types of target substances to be detected. They can be small molecule organic compounds, small molecule inorganic compounds, or high molecule organic compounds.

[0063] 4. Nucleic acid for response enhancement The nucleic acid used for response enhancement has the function of binding at least locally to a region complementary to the first nucleic acid, at least locally to a region complementary to the second nucleic acid, or at least locally to regions complementary to both (e.g., the regions of the first and second nucleic acids have palindromic structures on both sides). Similar to the nucleic acid aptamer beacons described above, the nucleic acid used for response enhancement can be DNA or RNA.

[0064] The length of the nucleic acid used for response enhancement is not particularly limited. However, from the perspective of having the function of complementary binding and competing with the complementary binding of the regions of the first and second nucleic acids, a certain length is preferred. For example, the length of the nucleic acid used for response enhancement can be 8 bases or more, 9 bases or more, or 10 bases or more. On the other hand, there is no particular upper limit to the length of the nucleic acid used for response enhancement. For example, the upper limit can be 50 bases or less, 25 bases or less, or 15 bases or less. Typically, it can be shorter than either the region of the first or second nucleic acid.

[0065] 5. Detection mechanism In one embodiment, the principle of detecting the target substance (60) in relation to the method of the present invention is explained. Figure 2 and Figure 1 Similar, but in terms of the presence of nucleic acids (70) for response enhancement. Figure 1 different.

[0066] and Figure 1 Similarly, the region (30) of the first nucleic acid and the region (40) of the second nucleic acid undergo complementary binding. This shortens the distance between the first substance (10) and the second substance (20) which are respectively linked to these regions. Therefore, the luminescence from the first substance (10) is modulated by the second substance (20). For example, when the second substance (20) functions as a quencher, the luminescence from the first substance (10) is extinguished.

[0067] However, in the presence of the target substance (60), the target substance (60) binds to the target substance binding region (50). This causes at least a localized structural change in the nucleic acid aptamer beacon. Furthermore, this structural change hinders the complementary binding of the region (30) of the first nucleic acid and the region (40) of the second nucleic acid. This increases the distance between the first substance (10) and the second substance (20). Therefore, the luminescence from the first substance (10) is not modulated by the second substance (20).

[0068] Here, the following two phenomena are in a state of equilibrium.

[0069] (1) The target substance (60) binds to the target substance binding region (50), and the region (40) of the second nucleic acid detaches from the region (30) of the first nucleic acid.

[0070] (2) The target substance (60) detaches from the target substance binding region (50), and the region (40) of the second nucleic acid binds to the region (30) of the first nucleic acid.

[0071] When the response-enhancing nucleic acid (70) binds complementary to the region (40) of the second nucleic acid, an equilibrium shift occurs in aspect (1) above. Then, by causing the equilibrium shift, the luminescence of the first substance (10) is enhanced. As a result, the detection sensitivity of the target substance (60) is improved.

[0072] Furthermore, in the example above, the response-enhancing nucleic acid (70) has the function of complementary binding to the region (40) of the second nucleic acid. In another example, the response-enhancing nucleic acid (70) has the function of complementary binding to the region (30) of the first nucleic acid. For example, the response-enhancing nucleic acid (70) has the function of complementary binding to a portion of the region (30) of the first nucleic acid.

[0073] As described above, when the target substance (60) binds to the target substance binding region (50), it causes a structural change that hinders the complementary binding of the region (30) of the first nucleic acid and the region (40) of the second nucleic acid. However, even if the hindered site is only a part of the region (30) of the first nucleic acid, the response-enhancing nucleic acid (70) may still bind complementaryly to the remaining part. Thus, it is possible to prevent the region (40) of the second nucleic acid from binding complementaryly to the region (30) of the first nucleic acid again.

[0074] 6. Other Furthermore, the terms "capable of complementary binding" and "functionality for complementary binding" used in this specification refer to the formation of a complex by two strands of nucleic acid. Typically, a complex is formed because the sequences of the two strands of nucleic acid are complementary. However, it is not necessary for the sequences of the two strands of nucleic acid to be completely complementary. For example, even if only one base pair out of 10 is not complementary, a complex can still be formed. Therefore, the terms "capable of complementary binding" and "functionality for complementary binding" not only include the case where the sequences of the two strands of nucleic acid are completely complementary, but also allow for cases where a small number of base pairs are not complementary. For example, within the entire region where complementary binding is intended to occur, non-complementary portions of less than 10% (e.g., only one base pair out of 10 is not complementary), less than 5%, or less than 1% are permissible.

[0075] Furthermore, the terms "capable of complementary binding" and "functionality to perform complementary binding" are not limited by specific conditions (e.g., salt concentration (i.e., ionic strength), temperature, etc.). As can also be understood from PCR (polymerase chain reaction), the two strands of nucleic acid separate or form a complex depending on temperature. In addition, sometimes the tendency for complex formation differs depending on the concentration of inorganic salts.

[0076] However, one embodiment of the method of the present invention relates to a method for detecting a target substance using a nucleic acid aptamer beacon. Therefore, it is necessary to detect the substance in the presence of the target substance. Thus, the terms "capable of complementary binding" and "functional for complementary binding" can indicate that the two strands can complementaryly bind under the conditions of the presence of the target substance or the conditions of detecting the target substance.

[0077] For example, in the case of detecting the presence of products in an enzyme reaction, the ability to perform complementary binding at the optimal temperature (e.g., 37°C) and optimal salt concentration of the enzyme reaction is included in the concepts of the terms "capable of complementary binding" and "functionality to perform complementary binding".

[0078] The term "nucleic acid" as used in this specification may include naturally occurring nucleic acids, or it may not include naturally occurring nucleic acids. Alternatively, the term "nucleic acid" as used in this specification may include chemically modified nucleic acids, or it may not include chemically modified nucleic acids. The terms "DNA" and "RNA" are used interchangeably in this specification. In addition, there is no particular limitation on the types of chemical modifications, but for example, it may include any one or more of the following: fluorescent labeling (e.g., FAM, TAMRA, ROX, etc.), methylation (e.g., methylation of 5-methylcytosine, methylation of the 2'-OH group of RNA, etc.), introduction of base analogs (e.g., 5-bromouracil (BrU), 6-thioguanine (6-TG), pseudouridine, etc.), fluorine modification (e.g., 2'-fluoroRNA (2'-F RNA)), introduction of linkers (e.g., PEGylation, biotin binding, etc.), poly(A) tail, morpholinonucleotide (PMO), peptide nucleotide (PNA), locked nucleotide (LNA), bridged nucleotide (BNA), unlocked nucleotide (UNA), glycerol nucleotide (GNA), thiophosphate ester bond, alkylphosphonate diester modification, etc.

[0079] 7. Testing Steps As described above, one embodiment of the method includes the following steps.

[0080] (A) The steps for mixing the target substance, nucleic acid aptamer beacon, and nucleic acid for response enhancement; and (B) The procedure for measuring the luminescence from the mixture.

[0081] Here, step (A) may include placing the target substance, the nucleic acid aptamer beacon, and the nucleic acid for response enhancement into the same container. More specifically, step (A) may also include placing the precursor of the target substance, the nucleic acid aptamer beacon, and the nucleic acid for response enhancement into the same container. In this case, the precursor of the target substance is converted into the target substance through a chemical reaction (including an enzymatic reaction, etc.). Then, the target substance generated through the chemical reaction can be detected.

[0082] Step (B) may include, for example, irradiating the mixture with light at or near the excitation wavelength of the first substance. Further, step (B) may involve measuring the luminescence from the second substance using an enzyme-linked immunosorbent assay (ELISA) reader, or measuring the effect of the second substance on extinction.

[0083] [Example]

[0084] Example 1 (Preparation of DNA Aptamer Beacons) In this embodiment, a DNA aptamer beacon (Ding and Liu, 2023) specifically for adenosine detection was used as a model. The DNA aptamer beacon consisted of a fluorescein (FAM)-labeled aptamer strand and a quencher (Black Hole Quencher1, BHQ1)-labeled strand (Table 1), both synthesized by Eurofins Genomics Co., Ltd. A solution of the DNA aptamer beacon was prepared by mixing 1 μM of the FAM-labeled aptamer strand, 2 μM of the quencher-labeled strand, 50 mM Tris-HCl (pH 7.5), 500 mM NaCl, and 20 mM MgCl2, heating at 95°C for 2 minutes, and then cooling to 25°C for 30 minutes to allow the two DNA strands to bind complementaryly. Three oligoDNAs (Oligo DNA-1, Oligo DNA-2, Oligo DNA-3) of different lengths complementary to the quencher-labeled strand (Table 1) were synthesized by Integrated DNA Technologies, Inc.

[0085] Table 1. Base sequences and modification sites of the oligoDNA used.

[0086] [Table 1] Oligomeric DNA Name Base sequence and modification sites FAM-tagged aptamer chain 5'-[FAM]CTCTCGACGACGTTTGCGATGAGAAACGTATGGTTTCGAAGGTCGTC-3' (Serial Number 1) Quenching agent marker chain 5'-AGTCGTCGAGAG[BHQ1]-3' (Serial Number 2) Oligo DNA-1 5'-CTCTCGACGACT-3' (Serial Number 3) Oligo DNA-2 5'-CTCTCGACGA-3' (Sequence Number 4) Oligo DNA-3 5'-CTCTCGAC-3' (Sequence Number 5)

[0087] Example 2 (Construction of adenosine standard curve using DNA aptamer beacons) Quantification of adenosine using DNA aptamer beacons was performed in polystyrene 96-well plates, and fluorescence was measured at 30°C using a microplate reader (Synergy H1, BioTek) (Ex 480nm, Em 530nm). Each well contained 100 μL of reagent, and the assay solution consisted of adenosine (various concentrations), DNA aptamer beacons (20 nM FAM-labeled aptamer strand), oligoDNA (OligoDNA-1, OligoDNA-2, OligoDNA-3) (various concentrations), 50 mM Tris-HCl (pH 7.5), 0.1 mM MgCl2, and 100 mM NaCl.

[0088] result To verify the effect of the quencher-labeled chain inhibiting the re-complementary binding of the FAM-labeled aptamer chain after dissociation, oligoDNAs of different lengths complementary to the quencher-labeled chain (Oligo DNA-1, Oligo DNA-2, and Oligo DNA-3) were added to the detection system, and their effects on the response to adenosine were analyzed. The results showed that 12-mer Oligo DNA-1 and 10-mer Oligo DNA-2 significantly improved the sensitivity of the nucleic acid aptamer beacon to adenosine, and this sensitivity improvement was more effective at longer base lengths and higher oligoDNA concentrations. Figure 3 ).

[0089] Based on the fluorescence intensity changes over time of adenosine addition using the aptamer beacon with oligoDNA (Oligo DNA-1), which showed the highest sensitivity enhancement effect, it was shown that the formation rate of the adenosine-FAM-labeled aptamer chain complex was increased by the addition of oligoDNA. Figure 4 This rate of increase depends on the concentration of oligomeric DNA.

[0090] To calculate the detection limit, lower limit of quantitation, and apparent dissociation constant of this detection system, a standard curve of adenosine over a wider concentration range was constructed. Figure 5 A). The detection limit (3.3σ / S) and the lower limit of quantitation (10σ / S) were calculated based on the standard deviation (n=10) of the fluorescence intensity (F0) of the blank value (0 μM SAM) and the slope (S) of the concentration range (0-2 μM) in the standard curve that showed a linear relationship (Table 2). Based on data from the adenosine standard curve ( Figure 5 A) Hanes-Woolf scatter plot ( Figure 5 The regression line of B) was used to calculate the apparent dissociation constant (Table 2).

[0091] Table 2. Effects of Oligo DNA-1 addition on detection limit, lower limit of quantitation, and apparent dissociation constant.

[0092] [Table 2] Oligo DNA-1 (0nM) Oligo DNA-1 (20nM) Oligo DNA-1 (80nM) Detection limit (μM) 1.38 1.22 0.511 Limit of quantification (μM) 4.19 3.70 1.55 Apparent dissociation constant (μM) 33.8 9.74 6.00

[0093] Based on these results, it was shown that the addition of Oligo DNA-1 (80 nM) reduced both the detection limit and the lower limit of quantitation by 63%, and the epigenetic dissociation constant by 82%. In this embodiment, oligomeric DNA that enhances the response of nucleic acid aptamer beacons through the addition of such DNA-1 is referred to as a response enhancing strand (RES, equivalent to the above-mentioned response enhancing nucleic acid).

[0094] Existing nucleic acid aptamer beacons, after SELEX screening, improve sensitivity or specificity by introducing local random mutations to optimize or enhance the binding site of the target molecule (Zhang et al., 2017). On the other hand, this invention uses a response-enhancing strand method that does not require modification of the nucleic acid aptamer itself. By designing and using a response-enhancing strand that can bind complementary to the base sequence exposed after interaction with the target molecule, the sensitivity reduction caused by re-complementary binding is suppressed, thereby achieving signal enhancement. This simple and modular strategy does not require the introduction of complex mutations as in existing methods and can be easily applied to existing nucleic acid aptamer beacons at low cost, thus it is believed to achieve efficient functional improvement.

[0095] The following documents were referenced in the above embodiments.

[0096] References (Non-patent literature) Ding Y, Liu J (2023) Pushing adenosine and ATP SELEX for DNA aptamers with nanomolar affinity. J Am Chem Soc 145: 7540-7547 Zhang Z, Oni O, Liu J (2017) New insights into a classic aptamer: binding sites, cooperativity and more sensitive adenosine detection. NucleicAcids Res 45: 7593-7601

[0097] The above describes specific embodiments of the invention. These embodiments are merely specific examples, and the invention is not limited to them. For example, the technical features disclosed in one embodiment can be applied to other embodiments. Furthermore, unless otherwise specified, the order of some steps in a particular method can be interchanged with other steps, and additional steps can be added between two specific steps. The scope of the invention is defined by the scope of the technical solution.

Claims

1. A method for detecting a target substance using nucleic acid aptamer beacons, characterized in that, The nucleic acid aptamer beacon has the following characteristics: (1) The region of the first substance emits fluorescence; (2) The region of the second substance, which modulates the luminescence produced by the first substance; (3) The region of the first nucleic acid, which is directly or indirectly connected to the first substance; (4) The region of the second nucleic acid, which is directly or indirectly linked to the second substance; and (5) The target substance binding region, which binds to the target substance and can cause structural changes in the nucleic acid aptamer beacon. The regions of the first nucleic acid and the second nucleic acid can be integrated, or they can exist separately. The regions of the first nucleic acid and the second nucleic acid have at least a portion of complementary sequences that can bind to each other. The method includes: (A) The step of mixing the target substance, the nucleic acid aptamer beacon, and the nucleic acid for response enhancement; and (B) The procedure for measuring the luminescence from the mixture. The nucleic acid for enhancing the response includes a region capable of complementary binding to a portion of a region of at least one of the regions of the first nucleic acid and the second nucleic acid.

2. The method according to claim 1, characterized in that, The first substance and the second substance are a combination of substances that cause the donor fluorescence to be extinguished by FRET.

3. The method according to claim 1, characterized in that, The first substance and the second substance are a combination of substances that induce fluorescent expression in the receptor via FRET.

4. The method according to any one of claims 1 to 3, characterized in that, The nucleic acid mentioned is DNA.

5. The method according to any one of claims 1 to 3, characterized in that, The nucleic acid in question is RNA.

6. The method according to any one of claims 1 to 3, characterized in that, The nucleic acid used for response enhancement has a base length of 8 or more bases.

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