Dual structure-switching aptamer-mediated signal amplification cascade system

Abstract

The present invention relates to a dual structure-switching aptamer-mediated signal amplification cascade system for virus detection. The system, according to the present invention, has the advantages of having a simple virus detection procedure and enabling specific detection even within a short detection time of 30 minutes or less, thereby not requiring expensive enzymes and thermal control equipment. In addition, the system has the advantage of enabling the detection of an actual pathogen (especially a virus) at a level of 1.0 PFU.

Description

Dual-structure-switching aptamer-mediated signal amplification cascade system

[0001] The present invention relates to a dual structure-switching aptamer-mediated signal amplification cascade system.

[0002] In late 2019, the first few cases of illness caused by a novel coronavirus, severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), were reported, and the virus quickly spread worldwide. The World Health Organization proposed naming the disease caused by SARS-CoV-2 coronavirus disease 2019 (COVID-19) and declared a pandemic on March 11, 2020. Unfortunately, as of February 13, 2023, more than 755 million cases of COVID-19 have been reported, 6.83 million of which have been fatal. New variants of SARS-CoV-2 have also emerged.

[0003] Molecular tests such as reverse transcriptase-polymerase chain reaction (RT-PCR) have been widely used to identify infectious diseases. While RT-PCR is widely used as the gold standard for detecting viral RNA (vRNA), it requires annealing procedures to resolve the stable structure of folded vRNA, as well as expensive equipment for temperature control and signal readout. To overcome these limitations, numerous isothermal amplification techniques have been reported for COVID-19 diagnosis that enable signal amplification without thermal regulation. These techniques include clustered regularly interspaced short palindromic repeats-Cas-based assays, loop-mediated isothermal amplification, hybridization chain reaction, catalytic hairpin assembly, and other hybridization tests. However, these amplification techniques often require multiple steps and high-purity RNA preparation for vRNA detection, limiting their widespread applicability in situations requiring rapid and direct detection.

[0004] Meanwhile, structure-switching aptamers are single-stranded DNA or RNA molecules engineered to incorporate a switching function. Standard aptamers are synthesized in vitro through a process called systematic evolution of ligands by exponential enrichment. This is advantageous because aptamers are chemically synthesized, making manipulation and chemical modification relatively simple compared to antibodies, and offering excellent reproducibility. Structure-switching aptamers are an attractive strategy because they eliminate the need to select entirely new aptamers and allow users to utilize aptamers that have been discovered and validated for affinity and specificity. The primary reason for using these converted aptamers is that the signal generated by target binding can be captured and used for target detection.

[0005] In addition to structure-switching aptamers, various Förster–Rhöddaud energy transfer (FRET)-based aptamer engineering strategies have been reported, such as aptamer beacons, split aptamers, and strand-displacement aptamers. Ellington and colleagues introduced the first structure-switching aptamer. Inspired by molecular beacons, they designed an aptamer beacon to detect thrombin molecules. The aptamer was designed to form a hairpin structure with complementary terminal regions at either end, each carrying a fluorophore and a quencher. Target binding triggers a conformational change in the aptamer beacon, resulting in increased fluorescence. Unlike aptamer beacons, which require direct modification of the aptamer sequence, strand-displacement structure-switching aptamers require minimal modification. These aptamers consist of a fluorophore-tagged aptamer and a short displaced strand with a complementary quencher tag attached to a region of the aptamer. Aptamer-target pairing allows the displacement of the short strand, resulting in fluorescence recovery. Therefore, structure-switching aptamers are intentionally designed to detect structural changes induced by target binding and thereby acquire signals. Based on this concept, several promising biosensors based on structure-switching aptamers have been developed, including colorimetric assays, chromatographic lateral flow assays, and electrochemical sensors. While structure-switching aptamer-based sensors are widely utilized for signal generation through target binding, their potential applications extend far beyond this function, and their range of applications is particularly broad.

[0006] One object of the present invention is to provide a composition for detecting a target protein comprising a dual-structure switching aptamer-mediated signal amplification cascade system.

[0007] Another object of the present invention is to provide a kit for detecting a target protein comprising the composition.

[0008] Another object of the present invention is to provide a method for detecting a target protein using a dual-structure switching aptamer-mediated signal amplification cascade system.

[0009] Another object of the present invention is to provide an information providing method for diagnosing target virus infection using a dual-structure switching aptamer-mediated signal amplification cascade system.

[0010] This is explained in detail as follows. Meanwhile, each description and embodiment disclosed in the present invention can also be applied to each other description and embodiment. In other words, all combinations of the various elements disclosed in the present invention fall within the scope of the present invention. Furthermore, the scope of the present invention should not be considered limited by the specific descriptions described below.

[0011] In addition, the terminology used in this specification is for the purpose of description only and should not be construed as limiting. The singular expression includes the plural expression unless the context clearly indicates otherwise. It should be understood that the terms “comprise” or “have” in this specification are intended to specify the presence of a feature, number, step, operation, component, part or combination thereof described in the specification, but do not exclude in advance the possibility of the presence or addition of one or more other features, numbers, steps, operations, components, parts or combinations thereof.

[0012] Additionally, unless otherwise defined, all terms used herein, including technical or scientific terms, have the same meaning as commonly understood by a person of ordinary skill in the art to which the embodiments pertain. Terms defined in commonly used dictionaries should be interpreted as having a meaning consistent with their meaning in the context of the relevant technology, and shall not be interpreted in an idealized or overly formal sense unless explicitly defined herein.

[0013] Furthermore, to avoid confusion due to overlapping content, the description of overlapping content has been omitted. In other words, the content of the invention is not limited to the content described below, and the content of the invention should be interpreted based on the overall content of the invention.

[0014] Numerous isothermal amplification techniques have been reported to enable signal amplification without thermal control for virus detection. These include clustered regularly interspaced short palindromic repeats-Cas-based assays, loop-mediated isothermal amplification, hybridization chain reaction, catalytic hairpin assembly, and other hybridization assays. However, these amplification techniques often require multiple steps and high-purity nucleic acid preparation for viral nucleic acid detection, limiting their widespread diagnostic applicability in situations requiring rapid and direct detection.

[0015] As a solution to the above-mentioned problem, the inventors of the present invention completed the present invention by developing a dual-structure switching aptamer-mediated signal amplification cascade system capable of rapidly and sensitively detecting viral proteins.

[0016] Accordingly, the present invention

[0017] 1) A first structural aptamer having the structure of the following general formula Ⅰ:

[0018] 5'-XYZ-3' (Ⅰ)

[0019] In the above general formula (Ⅰ),

[0020] The above X is an initiator moiety that has a hybridization sequence complementary to the linker polynucleotide of the second structure aptamer complex and induces an amplification cascade through hybridization with the linker polynucleotide of the second structure aptamer complex; the above Y is a spacer moiety for hairpin formation; the above Z is an aptamer moiety that binds to a target protein;

[0021] 2) A second structure aptamer complex having the following a), b) and c):

[0022] a) a linker polynucleotide having the structure of 5'-ABC-3',

[0023] In the above linker, A is a first pivot region, B is a sequence capable of hybridizing complementarily with the initiator portion of the first structure aptamer and also with a stopper polynucleotide, wherein the hybridization portion has a longer hybridization length for the stopper polynucleotide than the initiator portion of the first structure aptamer, and C is a sequence complementary to a sequence portion capable of binding to a dye.

[0024] b) a stopper polynucleotide capable of complementary binding to the B region of the above linker sequence, and

[0025] c) an aptamer having a sequence region that is complementary to the C region of the above linker sequence and is capable of binding to a dye,

[0026] 3) a fuel polynucleotide sequence capable of complementary binding to the linker through a second toehold generated by binding of the initiator of the first structural aptamer to the B region of the linker; and

[0027] 4) A composition for detecting a target protein including a dye is provided.

[0028] "Hybridization" in the present invention refers to the formation of a double-stranded nucleic acid from complementary single-stranded nucleic acids. Hybridization can occur when the complementarity between the two nucleic acid strands is perfect (a perfect match), or even when some mismatched bases are present. The degree of complementarity required for hybridization may vary depending on hybridization conditions, particularly temperature.

[0029] The term "target protein" refers to any type of protein to be detected, and may preferably be characterized by any type of viral protein, including structural proteins, non-structural proteins, regulatory and accessory proteins, and endogenous retroviral proteins, but is not limited thereto. According to one embodiment of the present invention, the protein may be a viral structural protein, such as a capsid protein or a viral envelope protein.

[0030] The above first structure aptamer may be a stem-loop structure that unfolds into a single strand in the presence of a target protein.

[0031] The present invention comprises two aptamers with structural switching capabilities and a fuel polynucleotide, whereby a cascade of strand hybridization and displacement triggers fluorescence generation and signal amplification. That is, when a target protein is present in a sample, the designed aptamers with structural switching capabilities generate fluorescence and amplify the signal through cascade strand hybridization and displacement.

[0032] The composition of the target protein detection composition according to the present invention is as follows, and a description of the design for the entire system is given in reference to Fig. 10c.

[0033] The X portion of the first structure aptamer has a stem-loop form before target protein binding, and the stem of the X portion of the first structure aptamer is preferably composed of 10 to 14 base pairs. To design an NBA (Anti-N protein structure-switching beacon aptamer), a careful balance between the self-hybridized stem-loop and the N-protein binding aptamer is required. An unstable stem-loop structure causes a decrease in the target-induced amplification cascade, and conversely, an overly stable stem-loop structure makes hairpin switching probabilistically impossible, thereby slowing down the target binding reaction. Therefore, the stem composed of 10 to 14 base pairs helps facilitate structure switching by providing appropriate stability to the aptamer.

[0034] The Z portion of the first structure aptamer is exposed when a target protein is present, thereby exposing the aptamer portion (Z) that binds to the initiator protein, and thus the target protein binds to the aptamer portion (Z) that binds to the protein. In other words, the Z portion of the first structure aptamer may bind to the target protein by being exposed by the conversion of the hairpin structure.

[0035] Additionally, the X site of the first structure aptamer is exposed by the conversion of the hairpin structure by target protein binding. This exposure induces the action of the X site as an initiator.

[0036] In the present invention, the term “transition of hairpin structure” means that the first structure aptamer is transformed from a hairpin form to a single-strand form.

[0037] In the present invention, the term “first structure aptamer” is used with the same meaning as “NBA (Anti-N protein structure-switching beacon aptamer)”.

[0038] The second structure aptamer complex is a form in which three structures, a stopper (b) and an aptamer (c) to which a dye is bound, are complementarily combined with a linker polynucleotide (a) as the center.

[0039] To facilitate the repetition of a reaction, a toehold is used to initiate the reaction. The term "toehold" according to the present invention refers to a sequence region that initiates a reaction via a complementary region. In other words, it refers to a sequence region that can be extended to have a sequence that is not complementary to the currently bound sequence.

[0040] The first toehold is preferably included in the linker polynucleotide and may consist of 5 to 10 nucleotides. It acts as a complementary sequence for binding to the X site of the first structure aptamer whose structure has been switched. The first toehold is a region that binds to the X site of the first structure aptamer that is exposed as the structure of the first structure aptamer is switched.

[0041] The C region of the above linker polynucleotide can exhibit luminescence by binding to a dye, which is released by the bond between the fuel polynucleotide sequence and the linker polynucleotide.

[0042] The second fulcrum is included in the linker polynucleotide. The second fulcrum is preferably present at a position having a longer hybridization length with respect to the stopper sequence than the initiator site of the first structure aptamer, and may preferably consist of 2 to 7 nucleotides. It has a sequence for binding to the fuel polynucleotide sequence and acts by binding to the fuel polynucleotide as a complementary sequence. That is, the second fulcrum is present in the B region of the linker polynucleotide and can complementarily bind to the fuel polynucleotide sequence by binding to the initiator site of the first structure aptamer.

[0043] At this time, the dye may be used without limitation as long as it is known as an aptamer-conjugated dye. For example, it may be at least one selected from among triphenylmethane dyes, such as crystal violet, methyl green, malachite green, eriogreen B, erioglosin A, and cetoglosin; DFHBI (3,5-difluoro-4-hydroxybenzylidene imidazolinone); TO (thiazole orange); and DFHO (4-(3,5-difluoro-4-hydroxybenzylidene)-1-methyl-5-oxo-4).

[0044] The fuel polynucleotide sequence may preferably consist of 30 to 36 base pairs. The binding affinity of the fuel-linker pair is known to affect the overall process because it is related to fluorescence generation. Therefore, when a fuel polynucleotide sequence consisting of the above number of base pairs is used, the optimal binding affinity can be exhibited.

[0045] In the present invention, the term “second structure aptamer complex” is used with the same meaning as “M-SDA (malachite green structure-switching strand displacement aptamer)”.

[0046] The process of detecting a target protein using the above composition is described in detail as follows.

[0047] The first structure aptamer (NBA) has a hairpin structure by a spacer region (Y), the target protein binding region (Z) is partially covered by the stem, and the initiator region (X) hybridizes with the spacer region and the target protein binding region in a stem-loop form.

[0048] 1) When a target protein is present, the first structure aptamer binds to the target protein by exposing the target protein binding site (Z) by converting the hairpin structure into a single-stranded form, thereby exposing the initiator site (X).

[0049] 2) The initiator portion (X) of the exposed first structure aptamer complementarily binds to the first fulcrum region (A) of the linker polynucleotide of the second structure aptamer complex (M-SDA), and thus the stopper polynucleotide bound to the linker is released, thereby generating the second fulcrum region.

[0050] 3) As the fuel polynucleotide sequence complementarily binds to the generated second toehold region, the first structure aptamer bound to the target protein and the aptamer (C) capable of binding to the dye are released.

[0051] At this time, the first structure aptamer bound to the released target protein binds to another second structure aptamer complex (M-SDA) and continuously releases an aptamer (C) capable of binding to a dye by repeating the reactions 2) to 3) above. This reaction continues until the second structure aptamer complex (M-SDA) and the fuel polynucleotide sequence are exhausted.

[0052] The aptamer capable of binding to the dye released in this manner can stabilize the planar structure of the dye by binding to it, thereby triggering color development (luminescence). Accordingly, the system according to the present invention can detect target proteins with extremely high sensitivity and specificity without the use of enzymes or temperature-controlled devices.

[0053] The present invention provides a kit for detecting a target protein comprising the composition for detecting the target protein described above.

[0054] The kit may preferably contain a fuel polynucleotide sequence at a concentration of 1 to 5 times that of the fuel polynucleotide sequence. As previously described, the system according to the present invention can be continuously used as long as the fuel polynucleotide is not depleted. Adding a fuel polynucleotide sequence at a concentration of 1 to 5 times can induce a rapid triggering of signal amplification. However, adding a fuel polynucleotide sequence at a concentration exceeding 5 times can cause an increase in noise, so adding an appropriate concentration of the fuel polynucleotide sequence is important.

[0055] Additionally, the kit may preferably include a dye at a concentration of 1 to 20 μM. Addition of a dye at a concentration exceeding 20 μM disrupts the system's balance and generates high noise, and thus, the best signal amplification efficiency can be achieved by adding a dye at a concentration of 1 to 20 μM.

[0056] Additionally, the kit can readily determine the optimal amount of reagents to be used in a particular reaction by those skilled in the art who have learned the disclosure herein. Typically, the kit of the present invention is manufactured in separate packages or compartments containing the aforementioned components.

[0057] Additionally, the kit may further include instructions for use and other tools or equipment necessary for detection. For example, the kit may further include a separate surfactant.

[0058] The present invention comprises (a) 1) a first structural aptamer having a structure of the following general formula Ⅰ:

[0059] 5'-XYZ-3' (Ⅰ)

[0060] In the above general formula (Ⅰ),

[0061] The above X is an initiator moiety that has a hybridization sequence complementary to the linker of the second structure aptamer complex and induces an amplification cascade through hybridization with the linker of the second structure aptamer complex; the above Y is a spacer moiety for hairpin formation; the above Z is an aptamer moiety that binds to a target protein;

[0062] 2) A second structure aptamer complex having the following a), b) and c):

[0063] a) a linker polynucleotide having the structure of 5'-ABC-3',

[0064] In the above linker, A is a first pivot region, B is a sequence capable of hybridizing complementarily with the initiator portion of the first structure aptamer and also with a stopper polynucleotide, wherein the hybridization portion has a longer hybridization length for the stopper polynucleotide than the initiator portion of the first structure aptamer, and C is a sequence complementary to a sequence portion capable of binding to a dye.

[0065] b) a stopper polynucleotide capable of complementary binding to the B region of the above linker sequence, and

[0066] c) an aptamer having a sequence region that is complementary to the C region of the above linker sequence and is capable of binding to a dye,

[0067] 3) a fuel polynucleotide sequence capable of complementary binding to the linker through a second toehold generated by binding of the initiator of the first structural aptamer to the B region of the linker; and

[0068] 4) A method for detecting a target protein is provided, including a step of reacting a dye with a sample.

[0069] A sample according to the present invention refers to any protein from a biological sample. The term "biological sample" refers to any sample containing a protein. The biological sample may be any tissue or body fluid obtained from a subject.

[0070] The biological sample includes, but is not limited to, sputum, blood, serum, plasma, blood cells (e.g., white blood cells), tissue, biopsy sample, smear sample, wash sample, swab sample, cell-containing body fluid, nucleic acid fluid, urine, peritoneal fluid and pleural fluid, cerebrospinal fluid, stool, tear fluid or cells therefrom of the subject. The biological sample may also include tissue sections taken for histological purposes, i.e., frozen or fixed sections or microdissected cellular or extracellular portions thereof. The biological sample may be obtained by a method that does not cause harm to the subject.

[0071] Preferably, the sample may be provided mixed in a buffer solution containing a surfactant.

[0072] In particular, the composition and / or kit according to the present invention has excellent detection efficacy, enabling detection even of very small concentrations of protein.

[0073] Accordingly, the above detection method may further include (c) a step of determining that the target protein has been detected when a fluorescence reaction is detected. The measurement of this fluorescence color change may be a conventional fluorescence measurement method in which the measurement wavelength of the fluorescence equipment is fixed and measured. Specifically, the measurement wavelength of the fluorescence equipment can be fixed and confirmed. For example, in the case of FAM fluorescence, a method in which the fluorescence intensity of the reactant at a wavelength of ex;495 / em;520 is measured and the fluorescence change is observed at intervals of 5 to 10 minutes for 1 to 2 hours can be used.

[0074] The present invention comprises: 1) a first structural aptamer having a structure of the following general formula Ⅰ:

[0075] 5'-XYZ-3' (Ⅰ)

[0076] In the above general formula (Ⅰ),

[0077] The above X is an initiator moiety that has a hybridization sequence complementary to the linker of the second structure aptamer complex and induces an amplification cascade through hybridization with the linker of the second structure aptamer complex; the above Y is a spacer moiety for hairpin formation; the above Z is an aptamer moiety that binds to a target protein;

[0078] 2) A second structure aptamer complex having the following a), b) and c):

[0079] a) a linker polynucleotide having the structure of 5'-ABC-3',

[0080] In the above linker, A is a first pivot region, B is a sequence capable of hybridizing complementarily with the initiator portion of the first structure aptamer and also a sequence capable of hybridizing with a stopper sequence, wherein the hybridization portion has a longer hybridization length with respect to the stopper sequence than the initiator portion of the first structure aptamer, and C is a sequence complementary to a sequence portion capable of binding to a dye.

[0081] b) a stopper polynucleotide capable of complementary binding to the B region of the above linker sequence, and

[0082] c) an aptamer having a sequence region that is complementary to the C region of the above linker sequence and is capable of binding to a dye,

[0083] 3) a fuel polynucleotide sequence capable of complementary binding to the linker through a second toehold generated by binding of the initiator of the first structural aptamer to the B region of the linker; and

[0084] 4) Provides a method for providing information for diagnosing viral infection, including a step of reacting a dye with a sample.

[0085] At this time, the above information provision method may further include (c) a step of determining that a virus has been infected when a fluorescence reaction is detected.

[0086] The virus may be, for example, any one selected from the group consisting of SARS-CoV-2 virus or a variant thereof, parainfluenza virus, parainfluenza virus type 2, parainfluenza virus type 3, influenza virus, influenza A virus, influenza B virus, adenovirus, rhinovirus, human immunodeficiency virus (HIV), tobacco mosaic virus (TMV), smallpox virus, poliovirus, foot-and-mouth disease virus, Ebola virus, lettuce mosaic virus, MERS virus, and Zika virus.

[0087] The system according to the present invention offers the advantages of a simple protein detection procedure and specific detection within a short detection time of less than 30 minutes, eliminating the need for expensive enzymes or thermal control equipment. In particular, the system can readily detect actual pathogens (especially viruses) even at low concentrations, making it ideal for use in the diagnosis of infectious diseases and other diseases.

[0088] Figure 1 is a schematic diagram showing the secondary structure of an anti-nucleocapsid (N) protein aptamer predicted using NUPACK software (http: / www.nupack.org).

[0089] Figure 2 shows the results of evaluating the binding affinity of anti-N protein aptamers NP-A48 and NP-A58 to N protein (a: SDS-PAGE gel electrophoresis results of wild-type (WT) and Omicron mutant N proteins, b: Western blot results for confirming N protein binding of NP-A48 and NP-A58).

[0090] Figure 3 is a schematic diagram showing the secondary structure of anti-N protein beacon aptamer (NBA) by stem length predicted using NUPACK software.

[0091] FIG. 4 is a result showing the design process of an anti-nucleocapsid (N) protein structure-switching beacon aptamer (NBA) according to the present invention (a: schematic diagram showing the composition of NBA and structure-switching through pairing with N protein, b: schematic diagram showing the engineering process from N protein aptamer to structure-switching aptamer using spacer and initiator sequences, c: result of confirming the binding affinity of NBA with 10-14 bp stem length to N protein of wild-type SARS-CoV-2 and Omicron variant).

[0092] FIG. 5 is a schematic diagram showing a fuel stimulus drive (FSP) amplification according to the present invention.

[0093] FIG. 6 shows the results of analyzing the fuel stimulator-driven (FSP) amplification product according to the present invention (a: amplification product PAGE gel image, b: amplification product fluorescence image; lane 1: FAM tag stopper 1 (FS), lane 2: stopper 2, lane 3: initiator (I), lane 4: linker (L), lane 5: fuel (F), lane 6: fuel-linker, lane 7: FS-linker, lane 8: (FS-linker)-fuel, lane 9: (FS-linker)-initiator, lane 10: FS-S2-linker, lane 11: FSP-fuel, lane 12: FSP-initiator, lane 13: FSP-initiator-fuel).

[0094] Figure 7 shows the results of measuring the fluorescence intensity of a 10 nm initiator, a 10 nm fuel, and an FSP solution containing both.

[0095] Figure 8 shows the results of confirming the binding process of the anti-nucleocapsid (N) protein structure switching beacon aptamer (NBA) and malachite green structure switching strand displacement aptamer (M-SDA) according to the present invention (a: Results of confirming fuel stimulant-driven (FSP) amplification products (lane 1: FAM tag stopper 1 (FS), lane 2: stopper 2 (S2), lane 3: linker (L), lane 4: fuel (F), lane 5: NBA, lane 6: FS-S2-L (FSP), lane 7: FSP-NBA, lane 8: FSP-NBA-N protein, lane 9: FSP-NBA-F, lane 10: FSP-NBA-N protein-F, lane 11: FSP-NBA-human serum albumin (HSA) and lane 12: FSP-NBA-HSA-F), b: Fluorescence intensity (FI) measurement results of FSP and NBA mixed solutions containing 1.0 ng N protein, 10 nm fuel DNA, and both; f: Comparison results of signal amplification efficiency according to stem length).

[0096] Figure 9 shows the results of the design process of malachite green (MG) structure-switching strand displacement aptamer (M-SDA) (a: Schematic diagram of fluorescence generation due to binding of released MG aptamer (MGA) and MG molecules, b: Evaluation of fluorescence generation due to strand displacement of MGA in linker, c: Polyacrylamide gel electrophoresis (PAGE) analysis of strand displacement reaction products, d: Fluorescence intensity (FI) measurement of M-SDA solution mixture containing 100 nm fuel and 100 nm initiator sequence together with fuel, e: Fluorescence intensity (FI) measurement of M-SDA and NBA solution mixture containing 100 nm fuel, 1.0 ng N protein, and both).

[0097] Figure 10 is a schematic diagram of a dual-structure switching aptamer-mediated signal amplification cascade for SARS-CoV-2 detection (a: Schematic diagram of collecting SARS-CoV-2 virion structure and clinical sample using nasopharyngeal swab; b: Schematic diagram of SARS-CoV-2 detection in clinical sample. Schematic diagram of chemically dissolving clinical sample in universal transport medium (UTM) and mixing with amplification cascade components and malachite green (MG); c: Schematic operation flow of dual aptamer-mediated signal amplification cascade).

[0098] Figure 11 shows the results of the optimization process of the dual-structure switching aptamer-mediated signal amplification cascade system according to the present invention (ab: signal amplification efficiency comparison results according to fuel length and signal-to-noise ratio (S / N ratio), cd: signal amplification efficiency comparison results according to fuel concentration and S / N ratio, ef: signal amplification efficiency comparison results according to malachite green (MG) concentration and S / N ratio). At this time, the signal-to-noise ratio was calculated based on the fluorescence intensity (FI) of the initiator per FI of NC (S / N ratio = initiator / FNC).

[0099] FIG. 12 shows the results of evaluating the sensitivity and selectivity of the dual-structure switching aptamer-mediated signal amplification cascade system according to the present invention (a: fluorescence intensity (FI) measurement results of the dual-structure switching aptamer-mediated signal amplification cascade system in the presence of various concentrations of N protein (10 fg to 1.0 ng), b: calibration curve of the dual-structure switching aptamer-mediated signal amplification cascade system, c: selectivity measurement results of the dual-structure switching aptamer-mediated signal amplification cascade system targeting various proteins).

[0100] FIG. 13 shows the results of evaluating the performance of a dual-structure switching aptamer-mediated signal amplification cascade system according to the present invention (a: results of measuring the fluorescence intensity (FI) of the system for various clades of wild-type SARS-CoV-2, b: results of measuring the fluorescence intensity (FI) of the system for various mutant SARS-CoV-2, c: results of measuring the fluorescence intensity (FI) of the system for various concentrations of SARS-CoV-2, D: results of measuring the fluorescence intensity (FI) of the system for various infectious viruses, e: results of confirming the detection performance of the system for nasopharyngeal clinical samples obtained from COVID-19 patients).

[0101] FIG. 14 shows the ROC (receiver operating characteristic) curves of the dual-structure switching aptamer-mediated signal amplification cascade system according to the present invention, tested on negative (n = 10) and positive (n = 25) nasopharyngeal swab samples.

[0102] The present invention will be described in more detail below through examples. However, these examples are intended to exemplify the present invention and the scope of the present invention is not limited to these examples.

[0103] Materials and Methods

[0104] 1. Reagent

[0105] 10X TBE (Tris-borate-EDTA) buffer and 1X TE (Tris-EDTA) buffer were purchased from BioWorld (Gyeonggi, Korea). Acrylamide / bis-acrylamide solution (19:1, 40%), tetramethylethylenediamine, stacking gel buffer, 10X Tris / glycine / SDS buffer, SDS, resolving gel buffer, and ammonium persulfate compound were purchased from Bio-Rad Laboratories (Hercules, CA, USA). Clinical virus transport medium was purchased from Noble Biosciences. Ultra-low range DNA ladder and PageRuler Prestained Protein ladder were purchased from Thermo Fisher Scientific (Waltham, MA, USA). Malachite green and magnesium chloride solutions were purchased from Sigma-Aldrich (St. Louis, MO, USA). Nuclease-free duplex buffer was purchased from Integrated DNA Technologies (Coralville, IA, USA). All oligonucleotides were purchased from Bioneer Co., Ltd. (Daejeon, South Korea), purified using high-performance liquid chromatography, and dissolved in TE buffer. Biotinylated aptamer sequences were synthesized using Integrated DNA Technologies. The specific sequences of all oligonucleotides used in this experiment are shown in Table 1.

[0106] [Table 1]

[0107]

[0108] 2. Virus

[0109] SARS-CoV-2 strains (NCCP nos. 43331, 43342, 43345, 43346, 43382, 43407, 43408, 43409), parainfluenza virus type 2 (NCCP no. 43178), parainfluenza virus type 3 (NCCP no. 43177), influenza A virus (NCCP no. 40214), influenza B virus (NCCP no. 42009), adenovirus (NCCP no. 40401), and rhinovirus (NCCP no. 40602) were provided by the National Center for Pathogen Cultures (NCCP) operated by the National Institute of Health. All experiments involving the use of viruses were conducted in biosafety level (BL-2 and BL-3) facilities at the Korea Research Institute of Bioscience and Biotechnology (KRIBB), approved by the Korea Centers for Disease Control and Prevention (KCDC), in compliance with institutional biosafety requirements. Before analysis, virus membranes were lysed with 0.5% TritonX-100. 2.5 μl of TritonX-100 was then added to 500 μl of virus samples and incubated at room temperature for 30 min.

[0110] 3. Clinical sample collection

[0111] Thirty-five nasopharyngeal samples were obtained from the Gyeongsang National University College of Medicine. Diagnostic results were compared with RT-qPCR results. The study protocol was reviewed and approved by the Institutional Review Board (IRB) of the Gyeongsang National University College of Medicine in Jinju (IRB Approval Number: 2022-10-012).

[0112] 4. Signal Amplification Cascade Protocol

[0113] Approximately 10.0 μl of virus sample was mixed with 10.0 μl of a 2X solution mixture containing 200 nm NBA (anti-N protein structure-switching beacon aptamer), 200 nm M-SDA (malachite green structure-switching strand displacement aptamer), 800 nm Fuel, and 30 μM MG (malachite green). The base buffer for this mixture was a 5x reaction buffer containing 250 mM Tris-HCl (pH 7.4), 50 mM NaCl, 700 mM KCl, and 50 mM MgCl2. The mixture was then incubated at room temperature for 30 min. After incubation, fluorescence intensity was measured using an Infinite M plex multimode plate reader (Tecan, Zurich, Switzerland) in a 384-well black flat-bottom polystyrene microplate (Corning, Corning, NY, USA).

[0114] 5. Fluorescence measurement

[0115] The fluorescence intensity of FSP in solution was observed using a Cytation 5 plate reader (Bio Tek, Winooski, VT, USA) at a fixed gain (λex = 484 nm and λem = 530 nm). The fluorescence intensity of MG in solution was observed using an Infinite M plex plate reader (Tecan) at a fixed gain (λex = 621 nm and λem = 665 nm).

[0116] 6. Electrophoretic analysis

[0117] The signal amplification cascade was confirmed using PAGE. The sequentially reacted oligonucleotides at each step were loaded onto the gel and dissolved in a 10% polyacrylamide solution containing 1X TBE buffer at a constant voltage (80 V) for 1 hour and 30 minutes at room temperature. The separated oligonucleotides were then stained with GelRed Nucleic Acid Gel Stain (Biotium, Inc., Fremont, CA, USA) for 10 minutes to visually confirm the FSP reaction using a gel imaging system (Bio-Rad Laboratories, USA).

[0118] 7. Western blotting

[0119] N proteins were separated using SDS-PAGE, transferred to polyvinylidene fluoride membranes (Invitrogen), and immunoblotted with the corresponding antibodies for each biotinylated aptamer. Immunodetection was performed using chemiluminescence after incubation with horseradish peroxidase-conjugated secondary antibody (Thermo Scientific).

[0120] 8. Data Analysis and Statistics

[0121] Data were obtained from at least three independent experiments. The exact number of experiments performed is included in each graph. Data were calculated as the mean (m) ± standard deviation (SD). The following legends were added to the figures: *p-value <0.05, **p-value <0.005, ***p-value <0.0005. The LOD was calculated using the following formula: LOD = 3σ / m, where σ is the standard deviation of the blank and m is the slope of the linear fit of the data in the linear range. ROC curves were estimated using MedCalc software (Ostend, Belgium).

[0122] Example 1. Development of a dual-structure switching aptamer-mediated signal amplification cascade system according to the present invention.

[0123] 1. Design of NBA (Anti-N protein structure-switching beacon aptamer)

[0124] We designed an anti-N protein structure-switching beacon aptamer (NBA) component to bind to the N protein, initiating a signal amplification cascade. The NBA is designed to have a stem-loop configuration. Upon binding to the N protein, the NBA undergoes a conformational change, exposing the initiator domain previously hidden within the stem-loop configuration.

[0125] Before designing the structural switching aptamer in earnest, two N protein aptamers, NP-A48 (SEQ ID NO: 1) and NP-A58 (SEQ ID NO: 2), were adopted and their binding affinities were compared, as shown in Fig. 1. The secondary structures of the aptamers were simulated using NUPACK software (http: / www.nupack.org).

[0126] As shown in Fig. 2, the binding of two aptamers, NP-A48 (SEQ ID NO: 3; biotin-linked to the 5' end of SEQ ID NO: 1) and NP-A58 (SEQ ID NO: 4; biotin-linked to the 5' end of SEQ ID NO: 2), to the N protein of the wild-type (WT) and Omicron mutants was confirmed through Western blot analysis. However, as shown in Fig. 2 a and b, the N protein aptamer NP-A48 was confirmed as a band at the correct position, whereas NP-A58 was not observed. Therefore, the NP-A48 aptamer was adopted for structural switching aptamer engineering.

[0127] Meanwhile, designing NBAs requires a careful balance between the thermodynamic equilibrium between the self-hybridized stem-loop and the N protein-binding aptamer. An unstable stem-loop structure results in a reduced target-induced amplification cascade. Conversely, an overly stable stem-loop structure probabilistically prevents hairpin switching, slowing down the target binding reaction.

[0128] Accordingly, as shown in Fig. 3, NBA was tested with stem lengths of 10, 12, and 14 bp.

[0129] As shown in Figure 4c, the strongest binding affinity was confirmed for a stem of 10 bp in length, and the longer the stem, the weaker the binding to the target.

[0130] However, since the detection sensitivity varies depending on whether an aptamer with high target binding affinity generates a high signal, we designed an M-SDA (malachite green structure-switching strand displacement aptamer) arbitrarily, confirmed the combination of M-SDA and each NBA, and then attempted to determine the length of the stem.

[0131] First, the components of M-SDA were designed based on fuel-stimulant-powered (FSP) amplification, a toehold-mediated strand displacement technique for signal amplification. As shown in Fig. 5, M-SDA (FSP probe complex) is composed of a linker (L; SEQ ID NO: 21), stopper 1 (S1; SEQ ID NO: 11), and stopper 2 (S2; SEQ ID NO: 13).

[0132] Specifically, the probe was designed to bind to the initiator of the switched NBA by including eight toehold sequences (the first toehold) in the linker. In the presence of the initiator, the stopper 2 region of the FSP probe is dissociated as the initiator displaces the stopper 2. This exposes five toehold sequences (the second toehold), inducing complementary hybridization between the linker and the fuel. Consequently, the initiator and stopper 1 regions dissociate from the linker and bind to another FSP probe.

[0133] As shown in Fig. 6a, the FSP amplification product was analyzed using PAGE. The gel image confirmed that Stopper 1 (SEQ ID NO: 11), Stopper 2 (SEQ ID NO: 13), and the linker sequence (SEQ ID NO: 21) formed an FSP complex (lane 10). Notably, the FSP complex remained stable even in the presence of fuel (lane 11). Furthermore, it was confirmed that the added initiator (SEQ ID NO: 20) induced the dissociation of Stopper 2 from the FSP complex (lane 12). Importantly, the fuel functions to generate waste, i.e., fuel-linker, and recycle the initiator (lane 13).

[0134] In addition, as shown in Fig. 6 b, the results of the FSP reaction were confirmed using the FAM-tagged stopper 1 (FS; SEQ ID NO: 12), and it was confirmed that the FS fragment was separated from the linker when fuel DNA was added in lane 13 of the fluorescence image.

[0135] Meanwhile, FRET-based validation was conducted using the aforementioned amplification cascade system. Quencher tag Stopper 2 (QS; SEQ ID NO: 14), which has a counter-receptor for FAM, was used to induce the FRET effect.

[0136] Figure 7 illustrates the function of the fuel used for signal amplification. Specifically, fluorescence intensity was observed after adding 10 nm fuel, initiator sequence, or both to a 10 nm FSP complex for 2 h. In the absence of initiator, no signal enhancement was observed. In contrast, when initiator was added to the solution, fluorescence intensity increased, indicating that the initiator replaced Stopper 2. Finally, recycling the initiator in the presence of both fuel and initiator resulted in a greater fluorescence increase.

[0137] Meanwhile, the NBA structure, which had undergone a structural transition upon binding to the N protein, enabled the initiation of a signal amplification cascade. As shown in Figure 8a, the NBA-N protein pair was demonstrated to bind to the FSP complex. Specifically, in the absence of the N protein, the NBA and FSP fragments did not bind to each other (lane 7). However, in the presence of the N protein, the NBA fragment disrupted the binding between the linker and the Stopper 2 fragment, displacing the Stopper 2 fragment and binding to the FSP fragment through the linker region (lane 8). The NBA and FSP probe complex remained stable even in the presence of fuel (lane 9). Furthermore, the cascade reaction occurred when the N protein and the fuel sequence were added together. This reaction generated the product, the linker-fuel complex, and displaced the Stopper 1 fragment from the linker (lane 10). In contrast, the cascade did not occur when human serum albumin (HSA) was added to the solution (lanes 11 and 12). The isolation of the Stopper 1 fragment indicates that the amplification cascade is activated by the binding of the fuel sequence, and fluorescence imaging confirmed that the FAM-tagged Stopper 1 (FS) fragment was displaced from the FSP complex.

[0138] Next, we verified whether this system enabled signal amplification. As shown in Figure 8b, fuel DNA and N protein were added alone or together to a mixture containing 10 nm NBA and 10 nm FSP, i.e., the FS-QS-linker complex, at concentrations of 10 nm and 1.0 ng, respectively, for 2 h. When fuel DNA alone was added to the mixture, no signal enhancement was observed compared to the negative control (NC). However, the fluorescence signal increased in the presence of N protein, indicating that the N protein-NBA fragment pair dissociated QS from the linker. Furthermore, the highest intensity was observed when both N protein and fuel DNA were present. These results indicate that the NBA fragment-N protein pair induces a conformational change in the FSP complex and can amplify the signal using fuel DNA.

[0139] Finally, as shown in Figure 8c, the three NBAs mentioned above were tested with various stem lengths to determine the optimal conditions. All NBAs showed enhanced fluorescence signals compared to the negative control (NC) in the presence of N protein, but the amplification efficiencies varied slightly. The fluorescence signal was increased 1.50-fold for S10 (SEQ ID NO: 5), 1.45-fold for S12 (SEQ ID NO: 7), and 1.30-fold for S14 (SEQ ID NO: 9). These results indicate that shorter stem lengths result in stronger binding to N protein. Overall, the S10 fragment, which exhibited the highest binding affinity to N protein, induced relatively high amplification efficiency.

[0140] 2. Design of M-SDA (malachite green structure-switching strand displacement aptamer)

[0141] To optimize the cascade system according to the present invention, an M-SDA structure was designed using multifunctional components responsible for signal generation and amplification. Specifically, unlike the above-mentioned M-SDA, an M-SDA was developed that allows for the binding of a malachit green aptamer (MGA) fragment to a linker instead of stopper 1 to generate a signal. As shown in Figure 9a, MGA was released from the M-SDA due to strand displacement caused by hybridization between the linker and the fuel. Before the MGA fragment was separated, it was partially bound to the linker, but after separation from the linker, it formed a specific secondary structure and formed a complex with the MG molecule.

[0142] Additionally, the fluorescent dye malachite green (MG) was treated with a solution containing MGA fragments and a solution containing the MGA-linker (MGA-L) complex. The MGA and MGA-L complexes were annealed at 95°C for 5 minutes and then slowly cooled to room temperature before use. Afterwards, the fluorescence intensity was measured to confirm the fluorescence generated due to strand displacement of MGA. As shown in Figure 9b, the fluorescence intensity of the MGA and MGA-L containing solutions added to the MG solution was no different from that of the MG solution alone. However, when fuel DNA was added, the fluorescence intensity of the MGA-L treated solution increased significantly. This increase in fluorescence intensity indicates that the MGA separated from the linker was folded into a secondary complex and bound to MG.

[0143] Additionally, strand displacement of MGA was investigated using PAGE after addition of fuel or initiator DNA, or both. The stopper was displaced (released) in the presence of the initiator fragment, and then the initiator was bound to the linker (lane 2, MGA-L-initiator complex). As shown in Figure 9c, in the absence of initiator, the M-SDA structure remained stable despite the presence of fuel DNA (lane 3). The MGA fragment was displaced (released) only when both initiator and fuel DNA were present together with M-SDA (lane 4).

[0144] Next, we observed the fluorescence intensity with and without the initiator, confirming that fluorescence generation was induced by the initiator sequence. As shown in Figure 9d, fluorescence intensity did not increase in the absence of the initiator sequence. In contrast, fluorescence increased dramatically when the initiator was added to the mixture.

[0145] Finally, the fluorescence intensity was measured after adding the NBA fragment and N protein to the M-SDA structure. As shown in Figure 9e, the NBA fragment-N protein pair slightly increased the fluorescence intensity, and the fuel DNA amplified the fluorescence signal through the cascade reaction. This enhancement in fluorescence intensity indicates that the designed dual-structure switching aptamer-mediated fluorescence amplification cascade functioned efficiently.

[0146] 3. Operation flow of the cascade system according to the present invention

[0147] A dual-structure switching aptamer-mediated signal amplification cascade according to the present invention was established for label-free detection of SARS-CoV-2. Specifically, all reaction steps for detection of the N protein of SARS-CoV-2 were performed simultaneously.

[0148] As shown in Figure 10a, SARS-CoV-2 consists of a single-stranded, positive-sense RNA genome surrounded by a lipid envelope comprising spike (S), envelope (E), and membrane (M) proteins, and the N protein. The N protein plays a crucial role in viral survival, including viral genome protection and host immune suppression. However, its high copy number per viral particle (~1,000 per particle) makes it an advantageous target for immunoassays.

[0149] Therefore, as shown in Figure 10b, a viral sample obtained using a patient's nasopharyngeal swab was chemically dissolved and directly mixed with the Cascade components in a tube. The resulting fluorescence was measured after adding the viral sample and incubating at room temperature for 30 minutes.

[0150] As shown in Fig. 10c, this system consists of four components: two structurally switching aptamers (NBA and M-SDA), fuel, and MG, and is performed in three stages: initiation, amplification, and fluorescence generation. The operating flow of the cascade system according to the present invention is described in detail as follows.

[0151] As shown in Figure 10c, the first step in SARS-CoV-2 detection is binding to the N protein and the NBA. The NBA (SEQ ID NO: 9) consists of three regions: an anti-N protein aptamer (red) for binding to the N protein of SARS-CoV-2, a spacer (orange) for hairpin formation, and an initiator (blue) that induces an amplification cascade through hybridization with M-SDA. The anti-N protein aptamer sequence (SEQ ID NO: 1) was hidden in the stem of the NBA, but when the N protein is present in solution, the structure of the NBA changes, allowing the aptamer sequence to bind to the N protein.

[0152] Meanwhile, the M-SDA component is the core of this diagnostic system and is designed to include functional segments involved in signal amplification and fluorescence generation. Specifically, as shown in Figure 10c, it consists of three DNA components: a linker (gray; SEQ ID NO: 21), a stopper (purple; SEQ ID NO: 13), and MGA (green; SEQ ID NO: 19). First, the linker contains an additional single-stranded sequence, namely toehold 1, for binding to the NBA-N protein pair. Among the M-SDA segments, the stopper controls nonspecific hybridization of the entire cascade process. The stopper regulates the reaction to operate in principle by preventing unconverted NBA from binding to M-SDA and fuel from binding to the linker. Finally, MGA generates fluorescence after being released from the linker.

[0153] The next step, amplification, can be triggered by fuel. When the stopper is released, toehold 2 opens, inducing complementary binding between the linker and the fuel. Consequently, the NBA-N protein pair dissociates from the linker and binds to another M-SDA. This cycle continues until the fuel is depleted.

[0154] The final step is to generate fluorescence. The isolated MGA, the final product of the amplification cascade, binds to a molecule called malachite green (MG), a widely used fluorescent aptamer-conjugated dye. When MG binds to MGA, the planar structure of this fluorescent dye is stabilized, resulting in fluorescence emission as the aptamer binding induces a red shift in the dye's absorbance. The generated signal can be helpful in identifying the N protein. Therefore, the system according to the present invention can enable sensitive SARS-CoV-2 detection without the use of enzymes or temperature-controlled devices.

[0155] Example 2. Optimization of a dual-structure switching aptamer-mediated signal amplification cascade system according to the present invention.

[0156] 1. System optimization

[0157] To optimize the amplification cascade, several tests were performed using initiators instead of N protein.

[0158] First, various fuel lengths were tested. The binding affinity of the fuel-linker pair is known to influence the overall process, as it is related to fluorescence generation. Therefore, fuels of 30-36 bp (SEQ ID NOs: 15-18) were designed and processed with M-SDA. As shown in Figure 11a, the fluorescence intensities of the NCs added to each fuel length in the mixture were measured similarly. As shown in Figure 11b, in the presence of an initiator, the highest fluorescence intensity and signal enhancement (signal-to-noise ratio, S / N) were observed for the 34 bp fuel (SEQ ID NO: 17). Therefore, 34 bp was selected as the fuel length for subsequent experiments.

[0159] Next, the fuel concentration was optimized. As shown in Fig. 11c, the fluorescence intensity increased up to 4 times the fuel concentration using the initiator, where nX represents the molar ratio of fuel to M-SDA. However, as the fuel DNA was added at higher concentrations, the fluorescence intensity of NC also showed a slight increase. The S / N ratio was confirmed to have no significant effect on the signal amplification up to a 3-fold increase in the fuel DNA concentration, but after a 4-fold concentration, the signal amplification increased rapidly. Higher fuel concentrations were tested, but the S / N ratio decreased due to increased noise (data not shown). Therefore, a 4-fold concentration was selected as the optimized fuel concentration.

[0160] Finally, the efficiency of signal amplification was investigated using various concentrations of MG. As shown in Figure 11e, excessive MG concentration disrupted the system's balance and generated high noise, necessitating optimization of the detection system. As shown in Figure 11f, the highest signal amplification efficiency was observed at a fuel concentration of 15 μM, and this concentration was used in future experiments.

[0161] 2. Evaluation of the sensitivity and selectivity of the system

[0162] The sensitivity of the system was measured under the previously established optimized conditions. As shown in Figure 12a, the fluorescence intensity was observed to gradually increase with increasing N protein concentration. Accordingly, the intensity was calculated to be linearly dependent on the logarithmic value of N protein concentration. As shown in Figure 12b, the linear fit equation was y = 37.086x + 555.53 (R2 = 0.9654).

[0163] Additionally, the limit of detection was calculated using the following formula: LOD = 3.3 × linear regression / slope standard deviation. The limit of detection for this system was estimated to be 5.87 × 10-15 g.

[0164] Meanwhile, system selectivity was confirmed by comparing the fluorescence intensities of several control proteins included in the mixture. Specifically, the system's affinity for control proteins, including spike protein (S, SARS-CoV-2), HSA, hexon protein (adenovirus), and fusion protein (respiratory syndrome virus), was tested.

[0165] As shown in Figure 12c, the fluorescence intensity of the control protein did not increase compared to the negative control (NC). These results indicate that the detection system exhibited high selectivity for the target protein.

[0166] Example 3. Performance Evaluation of a Dual-Structure Switching Aptamer-Mediated Signal Amplification Cascade System According to the Present Invention

[0167] The cascade system was applied using subtypes of SARS-CoV-2 identified in clinical patients. First, an attempt was made to detect four clades of wild-type SARS-CoV-2. As shown in Figure 13a, chemically lysed virus samples (10 4 When 100 PFU) was added to the mixture and the generated fluorescence was measured for 1 hour, the fluorescence intensity increased by at least 2,400 times compared to the negative control (NC).

[0168] Based on these results, SARS-CoV-2 variants, including beta, delta, omicron, and mu, were tested. As shown in Figure 13b, chemically dissolved 10 4 When the mutant samples of PFU were added to the mixture and the generated fluorescence was measured for 1 hour, it was found that the use of the mutants in the system resulted in a rapid increase in fluorescence intensity compared to the negative control (NC).

[0169] Next, the sensitivity of the cascade system to whole virus samples was verified. To this end, lysed virus samples were added for 15 minutes, after which fluorescence was measured. As shown in Figure 13c, the system according to the present invention was able to detect up to 1.0 PFU of SARS-CoV-2 within 30 minutes.

[0170] Additionally, the selectivity of the detection system for infectious viruses was also confirmed. As shown in Figure 13d, the system demonstrated stability even when used with other viruses, including human parainfluenza virus types 2 and 3, adenovirus, influenza A and B viruses, and human rhinovirus.

[0171] Finally, the system was tested using clinical samples. The clinical samples were obtained from the Gyeongsang National University College of Medicine. Fluorescence intensity was measured after 30 minutes of incubation at room temperature. As shown in Figure 13e, a total of 35 clinical samples were verified by quantitative RT-PCR (RT-qPCR), and overall, positive clinical samples showed higher signal intensity than negative samples.

[0172] Using these results, the receiver operating characteristic (ROC) curve was estimated. As shown in Figure 14, the area under the curve (AUC) of the system was 1.00. Using AUC as an indicator of predictive ability, we can conclude that the dual-structure switching aptamer-mediated signal amplification cascade can be used as a diagnostic tool.

Claims

1. 1) First structural aptamer having the structure of the following general formula Ⅰ: 5'-XYZ-3' (Ⅰ) In the above general formula (Ⅰ), The above X is an initiator moiety that has a hybridization sequence complementary to the linker polynucleotide of the second structure aptamer complex and induces an amplification cascade through hybridization with the linker polynucleotide of the second structure aptamer complex; the above Y is a spacer moiety for hairpin formation; the above Z is an aptamer moiety that binds to a target protein; 2) A second structure aptamer complex having the following a), b) and c): a) a linker polynucleotide having the structure of 5'-ABC-3', In the above linker, A is a first pivot region, B is a sequence capable of hybridizing complementarily with the initiator portion of the first structure aptamer and also with a stopper polynucleotide, wherein the hybridization portion has a longer hybridization length with respect to the stopper sequence than the initiator portion of the first structure aptamer, and C is a sequence complementary to a sequence portion capable of binding to a dye. b) a stopper polynucleotide capable of complementary binding to the B region of the above linker sequence, and c) an aptamer having a sequence region that is complementary to the C region of the above linker sequence and is capable of binding to a dye, 3) a fuel polynucleotide sequence capable of complementary binding to the linker through a second toehold generated by binding of the initiator of the first structural aptamer to the B region of the linker; and 4) A composition for detecting a target protein, comprising a dye.

2. A composition in the first paragraph, wherein the first structure aptamer is a stem-loop structure that is unwound into a single strand in the presence of a target protein.

3. A composition in the first paragraph, wherein the Z portion of the first structural aptamer binds to the target protein by being exposed by the conversion of the hairpin structure.

4. A composition according to claim 1, wherein the X portion of the first structural aptamer has a stem-loop form before binding to the target protein.

5. A composition in the fourth paragraph, wherein the stem of the first structure aptamer X portion is composed of 10 to 14 base pairs.

6. A composition according to claim 1, wherein the X portion of the first structural aptamer is exposed by conversion of the hairpin structure due to binding to the target protein.

7. A composition in the first paragraph, wherein the first toehold is a region that binds to the X site of the first structure aptamer that is exposed as the structure of the first structure aptamer is converted.

8. A composition according to claim 1, wherein the first toehold is composed of 5 to 10 nucleotides.

9. A composition according to claim 1, wherein the C region of the linker sequence is released by binding between the fuel polynucleotide sequence and the linker polynucleotide.

10. A composition according to claim 1, wherein the second toehold is a region that binds to a fuel polynucleotide sequence.

11. A composition according to claim 1, wherein the second toehold is composed of 2 to 7 nucleotides.

12. A composition according to claim 1, wherein the fuel polynucleotide sequence consists of 30 to 36 base pairs.

13. A kit for detecting a target protein comprising a composition for detecting a target protein selected from any one of claims 1 to 12.

14. A kit according to claim 12, wherein the kit comprises a fuel polynucleotide sequence at a concentration of 1 to 5 times.

15. A kit according to claim 12, wherein the kit comprises a dye at a concentration of 1 to 20 μM. 16.1) First structural aptamer having the structure of the following general formula Ⅰ: 5'-XYZ-3' (Ⅰ) In the above general formula (Ⅰ), The above X is an initiator moiety that has a hybridization sequence complementary to the linker polynucleotide of the second structure aptamer complex and induces an amplification cascade through hybridization with the linker polynucleotide of the second structure aptamer complex; the above Y is a spacer moiety for hairpin formation; the above Z is an aptamer moiety that binds to a target protein; 2) A second structure aptamer complex having the following a), b) and c): a) a linker polynucleotide having the structure of 5'-ABC-3', In the above linker, A is a first pivot region, B is a sequence capable of hybridizing complementarily with the initiator portion of the first structure aptamer and also with a stopper polynucleotide, wherein the hybridization portion has a longer hybridization length for the stopper polynucleotide than the initiator portion of the first structure aptamer, and C is a sequence complementary to a sequence portion capable of binding to a dye. b) a stopper polynucleotide capable of complementary binding to the B region of the above linker sequence, and c) an aptamer having a sequence region that is complementary to the C region of the above linker sequence and is capable of binding to a dye, 3) a fuel polynucleotide sequence capable of complementary binding to the linker through a second toehold generated by binding of the initiator of the first structural aptamer to the B region of the linker; and 4) dye; A method for detecting a target protein, comprising a step of reacting with a sample.

17. A method for detecting a target protein, further comprising the step of determining that the target protein has been detected when a fluorescence reaction is detected in the 16th paragraph (c). 18.1) First structural aptamer having the structure of the following general formula Ⅰ: 5'-XYZ-3' (Ⅰ) In the above general formula (Ⅰ), The above X is an initiator moiety that has a hybridization sequence complementary to the linker polynucleotide of the second structure aptamer complex and induces an amplification cascade through hybridization with the linker polynucleotide of the second structure aptamer complex; the above Y is a spacer moiety for hairpin formation; the above Z is an aptamer moiety that binds to a target protein; 2) A second structure aptamer complex having the following a), b) and c): a) a linker polynucleotide having the structure of 5'-ABC-3', In the above linker, A is a first pivot region, B is a sequence capable of hybridizing complementarily with the initiator portion of the first structure aptamer and also with the stopper polynucleotide, wherein the hybridization portion has a longer hybridization length for the stopper polynucleotide than the initiator portion of the first structure aptamer, and C is a sequence complementary to a sequence portion capable of binding to a dye. b) a stopper polynucleotide capable of complementary binding to the B region of the above linker sequence, and c) an aptamer having a sequence region that is complementary to the C region of the above linker sequence and is capable of binding to a dye, 3) a fuel polynucleotide sequence capable of complementary binding to the linker through a second toehold generated by binding of the initiator of the first structural aptamer to the B region of the linker; and 4) dye; A method for providing information for diagnosing a viral infection, comprising a step of reacting with a sample.

19. A method for providing information for diagnosing viral infection, further comprising the step of determining that infection with the target virus occurs when a fluorescence reaction is detected in paragraph 18.

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