Composition for detecting target nucleic acid and use thereof

Abstract

The present invention relates to a composition for detecting a target nucleic acid sequence and a nucleic acid detection method using same.

Description

Composition for detecting target nucleic acid and use thereof

[0001] The present invention relates to a composition for detecting a target nucleic acid and a nucleic acid detection method using the same.

[0002]

[0003] The foundation of modern molecular diagnostics is the precise detection of nucleic acids. Most current nucleic acid testing methods involve the use of biological components in an in vitro environment. For example, one of the most widely used techniques, polymerase chain reaction (PCR)-based testing, relies on the amplification of target nucleic acids using the DNA polymerase of thermophilic microorganisms. However, conventional PCR reactions require dozens of temperature adjustments to amplify target genetic material, requiring expensive equipment capable of reliably achieving high and low temperatures within a short period of time.

[0004] As an alternative to PCR, isothermal amplification methods have been studied. Isothermal amplification allows for amplification of target genetic material at a single temperature (room temperature or elevated temperatures below 65°C) without the need for changing the reaction temperature. This eliminates the need for expensive temperature control equipment, making it readily applicable to point-of-care diagnostics, which are difficult to implement with conventional PCR.

[0005] An example of an isothermal amplification method is the LAMP (Loop-Mediated Isothermal Amplification) reaction. The LAMP reaction is a reaction that amplifies DNA through a strand displacement reaction by creating a loop structure (stem-loop DNA) at the primer binding site from four primers that select six parts from the target DNA strand and combine them. It can bind to the target ssDNA and amplify it at an isothermal temperature of 60-65℃ (Nucleic Acids Res. 2000 Jun 15; 28(12): e63). However, the LAMP reaction has limitations in that it cannot work with short targets and can only detect nucleic acids consisting of a few hundred bases. Furthermore, the detection of nucleic acids amplified through PCR or isothermal amplification is limited to methods such as using double-stranded DNA-binding dyes such as SYBR Green or using probe sequences labeled with fluorescent dyes such as TaqMan technology. On the other hand, if a method is developed that detects the amplified transcription or translation product through transcription and translation reactions using the target nucleic acid instead of amplifying the target nucleic acid itself, it can be detected in various ways through the biological activity of the transcription or translation product, unlike existing methods with limited detection methods. For example, if transcription and translation reactions can be induced so that an enzyme protein is produced by the target nucleic acid, detection is possible using substrates that exhibit color or fluorescence through enzyme conversion, and analysis by the fluorescence emitted by the protein itself, such as green fluorescent protein (GFP), is also possible. Therefore, rather than detecting the target nucleic acid amplified through PCR or isothermal amplification methods as is currently used, if a technology is developed that can recognize the target nucleic acid and express an active protein from it, nucleic acid detection will be possible with greater ease and precision.

[0006]

[0007] As a result of our efforts to develop a new nucleic acid detection method, we developed a structure in which a target binding region annealing with a target sequence is connected to a single strand of a promoter at the upper end and a reporter nucleic acid or reporter protein coding sequence is connected at the lower end, and confirmed that by using this, signal generation through expression of a reporter nucleic acid or reporter protein is possible only when a target nucleic acid is present, thereby completing the present invention.

[0008]

[0009] An object of the present invention is to provide a composition for detecting a target nucleic acid comprising sensor DNA.

[0010] Another object of the present invention is to provide a method for detecting a target nucleic acid, comprising the steps of adding a sample to the composition; and performing at least one reaction among cell-free replication, transcription, and protein synthesis to measure the expression level of a reporter gene.

[0011] Another object of the present invention is to provide a use of a composition comprising sensor DNA for detecting target nucleic acids.

[0012]

[0013] Since the composition for detecting target nucleic acids of the present invention can easily detect nucleic acids in a sample, it can be usefully used in various fields including molecular diagnosis.

[0014]

[0015] Figure 1 relates to the preparation of a non-transcribable DNA template. Figure 1A shows a DNA template prepared by PCR using a uracil-containing forward primer, followed by treatment with USER (Uracil-Specific Excision Reagent) to expose a single-stranded TBS and promoter. Figure 1B shows the results of confirming the removal of the uracil segment by measuring fluorescence from a forward primer labeled with 5'-end Yakima-Yellow (YY) dye. The error bars in the graph represent the standard deviation of three independent experiments.

[0016] Figure 2 relates to oligonucleotide-triggered reporter protein synthesis. Figure 2A is a schematic diagram of the TASER assay. Figure 2B shows that sensor DNA containing a single-stranded T7 promoter is prepared by PCR using a uracil-containing primer followed by USER treatment, and that incubation of the sensor DNA alone with a cell-free protein synthesis reaction does not produce protein due to the absence of the double-stranded T7 promoter. The amount of sfGFP synthesized from various DNA templates was measured. Each sequence is, from top to bottom: no DNA, PCR results using dTTP, PCR results using a uracil-containing forward primer, and PCR results after USER treatment, which exposes the (-) strand of the T7 promoter and upstream sequences. Figure 2C shows that after annealing with an oligonucleotide containing a sequence complementary to the single-stranded T7 promoter, the sensor DNA becomes transcribable, thereby directing cell-free synthesis of the encoded protein. A double-stranded extension upstream of the T7 promoter significantly improved sfGFP synthesis efficiency. Circles in the graph represent signal-to-background ratios (S / B), and error bars represent the standard deviation of three independent experiments. T7P, T7 promoter; T7RNAP, T7 RNA polymerase.

[0017] Figure 3 shows the results of an agarose gel evaluation of RNA in vitro transcribed (IVT) from TASER sensor DNA. After the IVT reaction, a 1 μL aliquot was electrophoresed on a 1.5% agarose gel. RNA bands were visualized using SYBR Gold Nucleic Acid Gel Stain. Each IVT reaction contained: (A) a complete double-stranded PCR product; (BF) sensor DNA hybridized with each indicated target DNA; and (G) sensor DNA.

[0018] Figure 4 shows the results of a TASER assay confirming the activation of sensor DNA through annealing of target DNA to the upstream sequence of the T7 promoter. Figure 4A confirms the synthesis of sfGFP by a TASER assay using sensor DNA annealed to oligonucleotides covering the upstream sequence and various regions of the T7 promoter. Figure 4B is a schematic diagram of the TASER assay. When the target sequence anneals to the TBS (target nucleic acid binding sequence) upstream of the T7 promoter, nucleotide polymerization is driven by the PolI activity of the S12 extract used in the assay mixture, initiating nucleotide polymerization along the template. This enables the sensor DNA to subsequently transcribe and translate the encoded signal-generating enzyme. Figure 4C confirms the effect of supplementing the S12 extract with PolI and dNTPs. Figure 4D shows the results of agarose gel electrophoresis analysis of transcripts from sensor DNA annealed with T7UP(06 / 18) and T7UP(15 / 00) oligonucleotides. The effects of supplementing PolI and dNTPs were investigated. A 1.5% agarose gel containing 0.7 M formaldehyde was used, and RNA bands were stained with SYBR Gold Nucleic Acid stain. Figure 4E shows the expression efficiency of T7UP(15 / 00) annealed sensor DNA evaluated using the PURE system. Error bars represent the standard deviation of three independent experiments.

[0019] Figure 5 shows the results of a TASER assay confirming the effects of PolI and dNTP supplementation on reporter protein synthesis. Figure 5A shows the results of adding purified PolI at various concentrations to a TASER assay mixture containing 0.25 mM dNTP. Figure 5B shows the effects of additional dNTPs in a TASER assay, with the PolI concentration fixed at 0.2 U / μL. Error bars represent the standard deviation of three independent experiments.

[0020] Figure 6 shows the results of TASER analysis using various signal reading methods. Figure 6A shows the results of detecting the signal of target DNA through the color change of a substrate such as CPRG using β-galactosidase as a reporter protein. Figure 6B shows the results of detecting target DNA through luminescence using sensor DNA encoding NLuc. Figure 6C shows the results of confirming that target DNA can be easily detected by reading glucose produced by sucrose hydrolysis using sensor DNA encoding invertase with a personal glucose meter (PGM). Figure 6D shows the results of confirming that target DNA can be detected by a commercially available CDV antigen kit using sensor DNA encoding the nucleocapsid protein of CDV. Figure 6E shows the results of confirming that sensor DNA encoding sfGFP can detect target DNA by fluorescence measurement. Analyses were performed in the presence (+) or absence (-) of 500 nM T7UP(15 / 00) sequence as target DNA. Error bars represent the standard deviation of three independent experiments.

[0021] Figure 7 shows the results comparing the sensitivity of TASER assays using various reporter proteins. Measurements were performed using various T7UP (15 / 00) devices. Figure 7A shows the results of detecting the signal of target DNA through the color change of a substrate such as CPRG through a TASER assay using sensor DNA encoding β-galactosidase. Figure 7B shows the luminescence intensity generated in a TASER assay using sensor DNA encoding NLuc. Figure 7C shows the results of quantifying glucose produced by sucrose hydrolysis after a TASER assay using sensor DNA encoding invertase, measured using PGM. Band intensities were quantified using Image J software. Figure 7D shows the results of analyzing CDV antigen produced by target DNA through a TASER reaction using a lateral flow immunoassay kit. Band intensities were quantified using Image J software. Figure 7E shows the fluorescence intensity of a TASER assay using sensor DNA encoding sfGFP. Circles in the graph represent the S / B ratio, and error bars represent the standard deviation of three independent experiments. Data with a p-value less than 0.005 were considered statistically significant (*p-value<0.05, **p-value<0.01, ***p-value<0.005).

[0022] Figure 8 is a design of flap probes and invasive probes targeting CPV2a DNA sequences for cleavage mediated by FENI.

[0023] Figure 9 shows the results of FRET analysis confirming FENI-mediated trigger DNA generation. The invasive probe used for IA (Invasive Amplication) was labeled with FAM (6-carboxyfluorescein) at the 5' end and BHQ (Black Hole Quencher) at the junction with target DNA (CPV2a, 1 μM). FAM fluorescence released from BHQ after FENI-mediated IA reaction was quantified to track the release of cleaved flap sequences from the substrate DNA. Error bars represent the standard deviation of three independent experiments.

[0024] Figure 10 illustrates the integration of TASER assays and IA for detecting viral nucleic acid sequences. Figure 10A shows an IA-TASER assay for CPV2a ssDNA. First, a trigger DNA targeting the TBS of the sensor DNA is generated, and the trigger DNA is used in the TASER assay to express a reporter protein encoded by the sensor DNA. The results of the IA-TASER assay using 10 nM synthetic CPV2a ssDNA as the target DNA are shown in the presence and absence of 1.6 U / μL FENI. The sensor DNA encoding sfGFP was used here. Figure 10B shows the results confirming the differential detection of CPV2a (4408C) compared to FPV (4408A) using IA-TASER. The error bars represent the standard deviation of three independent experiments.

[0025] Figure 11 is a diagram illustrating the removal of background signals in IA-TASER analysis. Figure 11A shows the results confirming that, compared to the original TASER analysis, the IA-TASER analysis increased the background signal in the absence of target DNA, which affected the sensitivity. Figure 11B shows the hypothesis that the trigger sequence of the uncleaved flap probe anneals to the sensor DNA, resulting in the degradation of the 3' flap, and that the trigger sequence was extended by Pol I even in the absence of the target DNA sequence and FENI. Figure 11C shows the results of introducing a modification to the 3' end of the flap probe to reduce the generation of signals unrelated to the target sequence. In particular, it can be confirmed that the background signal was significantly reduced when a phosphorothioate bond was introduced to the terminal 5 nucleotides of the flap probe. Furthermore, the introduction of 2'-O-methoxy-ethyl (2'MOE) into the same polynucleotide effectively reduced the background signal, so combining these two modifications yielded a greater effect. When evaluated against a 10 nM target DNA sequence, the combined introduction of these modifications improved the S / B ratio from 2 to 22. Error bars represent the standard deviation of three independent experiments.

[0026] Figure 12 shows the optimization results of the IA-TASER analysis under various conditions. Figure 12A shows the results when 10 nM single-stranded DNA (ssDNA), 100 nM flap probe, and 100 nM invasion probe were reacted with various concentrations of FENI at 65°C. Figure 12B shows the results when 10 nM single-stranded DNA (ssDNA), 100 nM flap probe, and 100 nM invasion probe were reacted with 1.6 U / μL FENI at various temperatures. Figure 12C shows the results when 100 nM flap probe, 1.6 U / μL FENI, and various concentrations of invasion probe were reacted at 65°C. Figure 12D shows the results when 100 nM invasion probe, 1.6 U / μL FENI, and various concentrations of flap probe were reacted at 65°C. Each experiment was performed in the presence / absence (+ / -) of ssDNA. Circles within the graph represent the S / B ratio. Error bars represent the standard deviation of three independent experiments.

[0027] Figure 13 shows the results of measuring the fluorescence signal of synthesized sfGFP by varying the IA-TASER analysis time. The circles in the graph represent the S / B ratio. The error bars represent the standard deviation of three independent experiments.

[0028] Figure 14 illustrates an IA-TASER assay using sensor DNA encoding sfGFP, NLuc, and invertase. Figure 14A illustrates the IA-TASER assay process for detecting CPV2a DNA. Trigger DNA is generated from target single-stranded CPV2a DNA by the IA reaction. The generated trigger DNA then initiates the expression of reporter proteins such as sfGFP, NLuc, and invertase in the TASER assay. Figure 14B shows the measurement of the luminescence intensity of NLuc generated during the IA-TASER process. Figure 14C shows the measurement of the fluorescence intensity of sfGFP generated during the IA-TASER process. Figure 14D shows the results of PGM measurements of glucose generated from sucrose hydrolyzed by the invertase encoded by the sensor DNA. The circles in the graph represent the S / B ratio, and the error bars represent the standard deviation of three independent experiments.

[0029] Figure 15 shows a standard curve for LOD determination. Figure 15A shows the standard curve obtained from an experiment using a sensor DNA encoding sfGFP, and Figure 15B shows the standard curve obtained from an experiment using a sensor DNA encoding NLuc. The LOD was calculated using the formula 3σ / s, where σ is the standard deviation of the blank control and s is the slope of the standard curve. The error bars represent the standard deviation of three independent experiments.

[0030] Figure 16 illustrates the TASER principle of the present invention.

[0031] Figure 17A illustrates the structure of the TASER sensor DNA, Figure 17B illustrates the structure of the probe and sensor DNA used in the IA-TASER, and Figure 17C illustrates the operating principle of the IA-TASER.

[0032] Figure 18 shows the DNA production efficiency by flap endonucleases targeting ssDNA or RNA. Figure 18A shows the results of electrophoresis analysis using FEN1 or Tth DNA polymerase (Tth DNAP) in an IA reaction targeting 1 μM ssRNA or RNA. The reaction was performed at 60°C for 2 hours and visualized using SYBR Gold Nucleic Acid Gel Stain. Figure 18B shows the results of FRET analysis verifying FEN1 or Tth DNAP-mediated triggered DNA production. The FAM signal was measured at 10-minute intervals for 3 hours at 60°C.

[0033] Figure 19 shows the LOD (limit of detection) confirmation results for the IA-TASER assay targeting 16S rRNA. Figure 19A confirms the linear relationship between the target RNA concentration and the fluorescence signal. Figure 19B confirms the proportional relationship between the fluorescence signal and the log value of the target RNA concentration. The LOD was determined according to the 3σ / s formula. The error bars represent the standard deviation of three independent experiments.

[0034] Figure 20 shows the results of a multiplex detection of biological warfare agents (BWA). IA-TASER analysis was performed on rRNA from six pathogenic strains. Each target rRNA was present at a concentration of 100 nM. Origin2023b was used for heatmap data analysis.

[0035] Figure 21 shows the results of target specificity and efficiency verification of an IA probe set using a scrambled RNA mixture. Figure 21A shows the results of an IA-TASER analysis using probe sets each designed to target a cognate target RNA. The scrambled RNA mixture consisted of 16S rRNA from six pathogenic strains. The symbol (-) indicates the presence of a mixture of five RNAs excluding the target 16S rRNA for the probe used, while (+) indicates the inclusion of the target 16S rRNA (six types). The signal / background ratio in Figure 21B represents the ratio of signals observed when the target RNA for the probe is present compared to when the target RNA is absent.

[0036]

[0037] 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.

[0038] Furthermore, those skilled in the art will recognize or be able to ascertain, using no more than routine experimentation, numerous equivalents to the specific embodiments of the invention described herein. Furthermore, such equivalents are intended to be encompassed by the present invention.

[0039]

[0040] One aspect of the present invention provides a composition for detecting a target nucleic acid comprising sensor DNA.

[0041] In the present invention, "sensor DNA" refers to a nucleic acid structure that includes a target binding region capable of recognizing a target nucleic acid, a promoter, and a reporter gene linked downstream of the promoter, and that selectively enables expression of the reporter gene only in the presence of the target nucleic acid. The "sensor DNA" of the present invention may be used interchangeably with the terms "TASR (target-assisted synthesis of reporters)" or "TASER (target-assisted synthesis of enzyme reporters)".

[0042] The terms TASR and TASER can be used interchangeably even when the reporter gene encodes a reporter nucleic acid rather than a protein, such as a Broccoli aptamer or Spinach aptamer.

[0043] Specifically, the sensor DNA of the present invention may include the following structure:

[0044] Target nucleic acid sequence binding region - promoter - reporter gene

[0045] In the sensor DNA of the present invention, the target nucleic acid sequence binding region may be a single-stranded DNA (ssDNA) capable of annealing to the target nucleic acid sequence. Specifically, the single-stranded DNA capable of annealing to the target nucleic acid sequence may include a base sequence complementary to all or part of the target nucleic acid sequence.

[0046] Specifically, the single-stranded DNA capable of annealing to the target nucleic acid sequence may contain four or more bases. More specifically, the single-stranded DNA capable of annealing to the target nucleic acid sequence may contain twelve or more bases. For example, if the single-stranded DNA capable of annealing to the target nucleic acid sequence is composed of four to eleven bases, the target nucleic acid sequence may include a promoter sequence. However, if a reporter nucleic acid or reporter protein can be selectively expressed only in the presence of the target nucleic acid sequence, the length of the single-stranded DNA is not particularly limited thereto.

[0047] In the sensor DNA of the present invention, the promoter may be composed of a single strand.

[0048] In one embodiment, the reporter gene at the bottom of the promoter region included in the sensor DNA of the present invention may be in a double-stranded form.

[0049] More specifically, the sensor DNA of the present invention may comprise the following structure or a sequence complementary thereto:

[0050] (5') Target nucleic acid sequence binding region - T7 promoter - reporter gene (3')

[0051] In one embodiment, the single stranded region included in the sensor DNA of the present invention may be the antisense (-) strand in the structure.

[0052] The "T7 promoter" of the present invention is a promoter derived from T7 bacteriophage, and the T7 promoter sequence included in the sensor DNA of the present invention may be 5'-TAATACGACTCACTATA-3' or a complementary sequence thereto. However, the present invention is not limited thereto, and other promoter sequences and sequences complementary thereto, or sequences having the same function by adding, deleting, or substituting one or more bases may be used.

[0053] In one embodiment of the present invention, the sensor DNA has a structure in which the T7 promoter is single-stranded and transcription is not initiated by a polymerase, so that the reporter nucleic acid or reporter protein is not expressed in an environment in which the target nucleic acid does not exist. However, when the target nucleic acid exists, the target nucleic acid binds to the target nucleic acid sequence binding region, and then the T7 promoter below double-strands through an elongation reaction, thereby expressing the reporter nucleic acid or protein below the T7 promoter region.

[0054] More specifically, the sensor DNA of the present invention may include the structure disclosed in FIG. 17A, but is not limited thereto.

[0055] In one embodiment of the present invention, some of the sensor DNA exists as a single strand and some as a double strand, and to manufacture the sensor DNA, a uracil-specific cleavage reaction may be used, or a restriction enzyme and ligase may be used.

[0056]

[0057] Another aspect of the present invention is a composition for detecting a target nucleic acid comprising (i) sensor DNA, (ii) a flap probe, and (iii) FEN (flap endonuclease).

[0058] In one embodiment, the (i) sensor DNA

[0059] Trigger nucleic acid sequence binding region - promoter - reporter gene

[0060] Contains the structure of,

[0061] The above trigger nucleic acid sequence binding region and promoter are single-stranded DNA, and the reporter gene is double-stranded DNA.

[0062] The above (ii) flap probe

[0063] i) a trigger nucleic acid sequence and ii) a sequence capable of annealing to a target nucleic acid sequence,

[0064] The above flap probe is characterized in that, when a target nucleic acid sequence exists, ii) a sequence capable of annealing to the target nucleic acid sequence is annealed with the target nucleic acid sequence, and the probe nucleic acid sequence and the target nucleic acid sequence form a non-complementary flap structure, so that i) the trigger nucleic acid sequence is cleaved by FEN.

[0065] That is, when a nucleic acid sequence to be detected (target nucleic acid sequence) exists, the trigger nucleic acid sequence included in the flap probe is cleaved by FEN and binds to the sensor DNA, thereby enabling expression of the reporter gene included in the sensor DNA.

[0066] A sequence capable of annealing to a target nucleic acid sequence may include a base sequence complementary to all or part of the target nucleic acid sequence.

[0067] In one embodiment, the flap probe may have the structure of i) at the 5' position and the structure of ii) downstream therefrom.

[0068] In any one of the aforementioned embodiments, the flap probe may comprise a modification at the downstream end of the sequence capable of annealing to the target nucleic acid sequence that prevents degradation of the flap. In any one of the aforementioned embodiments, the flap probe may comprise a modification at the 3' end that prevents degradation of the flap.

[0069] The modification that prevents the degradation of the above flap may be a modification that prevents a non-specific reaction in which the flap portion of the flap probe binds to the trigger nucleic acid sequence of the sensor DNA and then the 3' end is removed by a nuclease and then extended, thereby expressing the reporter gene even when the target nucleic acid is not present.

[0070] For example, the modification may include chemically modifying or altering the 3'-terminal base of the flap probe sequence, for example, modification with 2'-O-methoxy-ethyl (2'MOE), and / or phosphorothioate (pt).

[0071] In any one of the embodiments described above, the flap probe may have a structure of 5' - (trigger nucleic acid sequence) - (sequence capable of annealing to a target nucleic acid sequence) - 3', and may include a modification at the 3' end that inhibits flap degradation.

[0072] In any one of the embodiments described above, the composition for detecting a target nucleic acid may further comprise a probe capable of binding to the target nucleic acid. The additionally included probe may be referred to as an invasive probe, and the region where the invasive probe binds to the target nucleic acid may be adjacent to the region where the flap probe binds to the target nucleic acid, thereby forming a structure in which the structure of flap probe-target nucleic acid sequence-invasive probe is recognized and cleaved by a nuclease.

[0073] In any one of the embodiments described above, the composition for detecting the target nucleic acid may include a sensor DNA, an invasion probe, a flap probe, and a FEN as disclosed in FIG. 17B.

[0074] In the present invention, FEN (flap endonuclease) is an enzyme having flap cleavage activity, and includes an enzyme having endonuclease activity that recognizes and cleaves a flap sequence at a junction between double-stranded DNA and single-stranded DNA, and an enzyme having endonuclease activity that recognizes and cleaves a flap sequence of an RNA-oligonucleotide. The FEN may be, for example, FENI derived from Pyrococcus furiosus or DNA polymerase derived from Thermus thermophilus (Tth).

[0075] In one embodiment, when the target nucleic acid is a DNA sequence, the FEN may be FENI derived from Pyrococcus furiosus.

[0076] In one embodiment, when the target nucleic acid is an RNA sequence, the FEN may be a DNA polymerase derived from Thermus thermophilus (Tth).

[0077] In one specific embodiment of the present invention, when a newly designed TASER sensor DNA is manufactured to include a target nucleic acid binding region, it was confirmed that there is a hassle in that a sensor DNA including a structure in which a single strand is newly exposed must be manufactured every time the target sequence is changed. To solve this problem, the sequence exposed as a single strand is fixed to a sequence complementary to the trigger nucleic acid sequence, and only the target nucleic acid binding sequence of the probe is changed according to the target nucleic acid, thereby designing it so that detection of various target nucleic acids can be performed using only one common sensor DNA. That is, as shown in FIGS. 17B and 17C, the probe sequence forms a flap structure in the presence of the target nucleic acid sequence, is cleaved by FENI to generate a common trigger DNA, and this binds to the sensor DNA, thereby expressing the reporter gene included in the sensor DNA.

[0078] In another specific embodiment of the present invention, a trigger nucleic acid sequence cleaved by the FENI in the presence of a target nucleic acid sequence is amplified by PCR and reacted with sensor DNA, thereby enabling highly sensitive detection.

[0079] In another specific embodiment of the present invention, in order to prevent the generation of a background signal by binding of a flap probe and a sensor DNA without a cleavage reaction by FEN, the 3' end of a flap probe having the structure of (5')-trigger nucleic acid sequence - sequence capable of annealing to a target nucleic acid sequence - (3') is modified with phosphorothioate bonding and 2'MOE to prevent the degradation of the 3' flap, thereby enabling target nucleic acid-specific detection.

[0080]

[0081] In the present invention, a “reporter gene” or “reporter nucleic acid” is a DNA or RNA sequence that is replicated or transcribed from sensor DNA and generates a signal whose production or synthesis can be easily confirmed.

[0082] Specifically, the reporter nucleic acid may be an aptamer. For example, the aptamer may have a label attached to it. For example, the label may be a fluorescent substance, a radioisotope, a light-emitting element, an enzyme, or a nanoparticle. However, the present invention is not limited thereto.

[0083] Specifically, the reporter nucleic acid may be selected from the group consisting of, but is not limited to, a malachite green aptamer that generates fluorescence by binding to malachite green dye, (5Z)-5-[(3,5-Difluoro-4-hydroxyphenyl)methylene]-3,5-dihydro-2,3-dimethyl- 4H-Imidazol-4-one, a broccoli aptamer or spinach aptamer that generates fluorescence by binding to (Z)-4-(3,5-Difluoro-4-hydroxybenzylidene)-1,2-dimethyl-1H-imidazol-5(4H)-one (DFHBI), a mango aptamer, and a BFR (Blue Fluorescent RNA) aptamer.

[0084] In any of the embodiments described above, the reporter gene of the present invention may encode a reporter protein. In the present invention, the term "reporter protein" refers to a marker protein that is replicated or transcribed from sensor DNA and generates a signal that can easily detect its production or synthesis. The signal may take various forms, such as luminescence, fluorescence, phosphorescence, color development, or electron transfer.

[0085] Specifically, the reporter protein may be selected from, but is not limited to, sfGFP (superfolder green fluorescence protein), GFP (Green fluorescent protein), YFP (Yellow fluorescent protein), RFP (Red fluorescent protein), mCherry fluorescent protein, invertase, lactamase, galactosidase, HRP (Horseradish peroxidase), glucose oxidase, and luciferase. Among the above proteins, the production or synthesis of fluorescent proteins can be confirmed by measuring the fluorescence of the fluorescent protein accumulated in the cell-free synthesis reaction solution. In the case of enzymes, the synthesis of the reporter protein is confirmed by measuring the production level of the product using a substrate corresponding to each enzyme.

[0086] For example, the reporter protein may be a fluorescent protein or luciferase, such as sfGFP, firefly luciferase (FLuc), or deep-sea shrimp luciferase (NLuc).

[0087] As another example, the reporter protein may be an antigen protein. The antigen protein may be one that can be detected by a commercially available kit, and an example thereof may be the nucleocapsid protein of canine distemper virus (CDV Ag, GenBank accession number: AAC26990.1).

[0088] However, without limitation to the examples described above, any protein or nucleic acid sequence that generates a signal whose production or synthesis can be easily confirmed is included without limitation.

[0089]

[0090] In the present invention, "target nucleic acid" refers to the nucleic acid to be detected. The target nucleic acid sequence may be DNA or RNA, and may include not only ssDNA and RNA, but also dsDNA sequences. dsDNA can be used by switching the complementary strands. For example, ssDNA can be separated and used, or ssDNA can be exposed and used.

[0091] The length of the target nucleic acid sequence is not particularly limited, as long as the sensor DNA of the present invention selectively expresses a reporter gene only in the presence of the target nucleic acid sequence. For example, if the sensor DNA directly includes a target nucleic acid binding region, the length of the target nucleic acid sequence may be 12 nt or more, for example, 15 nt or more. If the sensor DNA directly includes a target nucleic acid binding region and the target nucleic acid sequence includes a T7 promoter sequence, the length excluding the T7 promoter sequence may be 4 nt or more. However, this is not limited thereto.

[0092]

[0093] As another example, if the composition provided by the present invention includes a flap probe and the sensor DNA does not directly include a region that binds to a target nucleic acid to be detected, but includes a trigger nucleic acid sequence, the length of the trigger nucleic acid sequence may be 12 nt or longer, for example, 15 nt or longer, but is not necessarily limited thereto.

[0094]

[0095] The composition of the present invention may further include any components necessary for nucleic acid and protein synthesis. Specifically, the components may be components necessary for one or more of cell-free replication, cell-free transcription, and cell-free protein synthesis.

[0096] In the present invention, "cell-free replication / transcription / protein synthesis" means performing replication / transcription / protein synthesis, which is performed inside cells, outside the body, such as in vitro. For example, cell-free protein synthesis means extracting only the components necessary for protein production, i.e., the intracellular protein synthesis machinery and its factors, from the cell, and artificially repeating only the protein synthesis process outside the cell while excluding the physiological control mechanism of the cell, thereby producing the target protein in a short period of time. At this time, the protein biosynthetic machinery required for cell-free protein synthesis, i.e., ribosome, initiation factor, elongation factor, termination factor, aminoacyl tRNA synthetase, RNA polymerase, etc., can be used as contained in the cell extract, added separately, or produced separately using genetic recombination technology. Similarly, cell-free replication means extracting the components necessary for nucleic acid replication from the cell and performing nucleic acid replication outside the cell.

[0097] In one embodiment, the composition of the present invention may include a component included in an E. coli crude extract. In one embodiment, the composition of the present invention may include any component necessary for DNA repair. In one embodiment, the composition of the present invention may include a DNA polymerase such as DNA polymerase I (Pol I). An example thereof may be E. coli-derived DNA polymerase I. In one embodiment, the composition of the present invention may include NTPs or dNTPs. In one embodiment, the composition of the present invention may include T7 RNA polymerase. However, the component included in the composition of the present invention is not particularly limited as long as it selectively replicates / transcribes or expresses a reporter gene only in the presence of a target nucleic acid sequence.

[0098] In one embodiment of the present invention, a structure of a sensor DNA was designed in which a target binding region in the form of ssDNA that anneals to a target sequence is connected to a single strand of a T7 promoter, and a reporter nucleic acid sequence (aptamer) or a protein coding sequence is connected to the lower strand, and in the presence of an E. coli crude extract, it was confirmed that the sensor DNA can selectively express a reporter nucleic acid or reporter protein only in the presence of the target sequence, and thus it was confirmed that it can be used for the detection of a target nucleic acid. In addition, when the sensor DNA is expressed in a PURE system that uses purified protein synthesis machinery instead of a crude extract that already contains DNA Pol I and dNTPs necessary for DNA repair, it was confirmed that the sensor DNA can selectively synthesize a reporter protein only in the presence of a component to which DNA Pol I and dNTPs are separately added, and thus it was confirmed that it can be used for the detection of a target nucleic acid.

[0099]

[0100] Another aspect of the present invention provides a method for detecting a target nucleic acid, comprising the steps of: adding a sample to the composition for detecting a target nucleic acid of the present invention; and performing at least one reaction among cell-free nucleic acid replication, transcription, and protein synthesis to measure the expression level of a reporter gene.

[0101] The composition for detecting the target nucleic acid, the reporter gene, and the cell-free protein synthesis reaction are as described above.

[0102] In the above method, when using a composition for detecting a target nucleic acid comprising (i) sensor DNA, (ii) a flap probe, and (iii) FEN (flap endonuclease), the method may include a step of reacting the flap probe and the sample in the presence of FEN; a step of PCR amplifying the nucleic acid present in the reaction product; and a step of reacting the amplified PCR product with the sensor DNA.

[0103] In the step of measuring the expression level of the reporter gene, methods known in the art can be used. For example, if the reporter protein is a fluorescent protein or the reporter nucleic acid is a fluorescently labeled aptamer, the expression of the reporter gene can be confirmed by measuring fluorescence.

[0104]

[0105] In the present invention, the sample refers to a sample to which the target nucleic acid detection method of the present invention is applied, which contains or does not contain a target nucleic acid to be detected.

[0106] As an example of an embodiment, the sample may be derived from one or more selected from the group consisting of, but not limited to, feed, food or chemicals.

[0107] In one embodiment, the sample may be isolated from a living organism. In one embodiment, the living organism includes both a plant and an animal.

[0108]

[0109] As an example of an implementation of the target nucleic acid detection method of the present invention, the target nucleic acid may be a nucleic acid sequence of a virus. In this case, the sample may be isolated from an individual suspected of being infected with the virus.

[0110] For example, the target nucleic acid may be a nucleic acid derived from a DNA or RNA virus.

[0111] For example, the target nucleic acid may be a nucleic acid derived from an ssDNA or dsDNA virus.

[0112] The above virus is not limited as long as its genome can be detected by the target nucleic acid detection method of the present invention. For example, parvovirus (e.g., canine parvovirus (CPV), feline parvovirus (FPV), human papillomavirus (HPV), polyomavirus, adeno-associated virus (AAV), parvovirus B19, human bocavirus, buphavirus, human parv4 G1), hepadnavirus (e.g., hepatitis B virus (HBV)); herpesvirus (e.g., herpes simplex virus (HSV), varicella-zoster virus (VZV), Epstein-Barr virus (EBV), cytomegalovirus (CMV), herpes lymphotropic virus, pityriasis rosea, Kaposi's sarcoma-associated herpes virus); Adenoviruses (e.g., atadenovirus, abiadenovirus, itaadenovirus, mastadenovirus, cyanadenovirus); poxviruses (e.g., variola, vaccinia virus, vaccinia virus, monkeypox virus, orf virus, pseudovaccinia, bovine papular stomatitis virus; tanapoxvirus, yabbai monkey tumor virus; molluscum contagiosum virus (MCV)); morbilliviruses (e.g., canine distemper virus), Geminiviridae; Nanoviridae; Phycodnaviridae, etc.), but are not limited thereto.

[0113]

[0114] As another example of the target nucleic acid detection method of the present invention, the target nucleic acid may be an RNA nucleic acid derived from an RNA virus.

[0115] In another embodiment of the target nucleic acid detection method of the present invention, the target nucleic acid may be a 16S rRNA sequence of a microorganism. Examples thereof include, but are not limited to, E. coli, Bacillus anthracis (B. anthracis), Francisella tularensis (F. tularensis), Yersinia pestis (Y. pestis), Burkholderia pesudomallei (B. pseudomallei), and Brucella abortus (B. abortus).

[0116] Hereinafter, the present invention will be described in more detail through examples and experimental examples. However, these examples and experimental examples are intended to exemplify the present invention, and the scope of the present invention is not limited to these examples and experimental examples.

[0117]

[0118] Example 1. Development of TASER and IA-TASER

[0119] ingredient

[0120] ATP, GTP, UTP, CTP, creatine phosphate, creatine kinase, and E. coli MRE600 total tRNA mixture were purchased from Roche Applied Science (Indianapolis, IN, USA). Target DNA oligonucleotides (<100 nucleotides) and PCR primers were synthesized by Integrated DNA Technologies (Coravile, Iowa, USA). High-fidelity VELOCITY DNA polymerase and Phusion U Hot Start DNA polymerase were purchased from Bioline (London, UK) and Thermo Fisher Scientific (Waltham, MA, USA), respectively. E. coli polymerase I, uracil-specific excision reagent (USER), and Xba1 restriction enzyme were purchased from New England Biolabs (Ipswich, MA, USA). T4 DNA ligase was purchased from SolGent Co., Ltd. (Daejeon, Korea). Other reagents were purchased from Sigma-Aldrich (St Louis, MO, USA) and used without further purification. Nano-Glo luciferase assay kit, RiboMAX large-scale RNA production system-T7, and PCR clean-up kit were purchased from Promega (Madison, WI, USA). RNeasy RNA purification kit was purchased from Qiagen (Hilden, Germany). 96-well microplates were purchased from SPL Life Sciences (Pocheon, Korea). BL21Star (DE3) strain was obtained from Invitrogen (Waltham, MA, USA).

[0121]

[0122] Preparation of sensor DNA and target DNA

[0123] The sensor DNA was designed to recognize a target nucleic acid sequence and catalyze the synthesis of a signal-generating protein. The following proteins were used as the proteins encoded by the sensor DNA: superfolder green fluorescence protein (sfGFP), bacterial invertase (EC 3.2.1.26), β-galactosidase from Escherichia coli (β-gal; EC 3.2.1.23), nucleocapsid protein of canine distemper virus (CDV Ag, GenBank accession number: AAC26990.1), and NLuc. The reporter gene in the sensor DNA was designed to be regulated by the T7 promoter. That is, since the upstream sequence containing the T7 promoter in the sensor DNA is single-stranded and only the protein-coding region of the sensor DNA is double-stranded, protein expression by itself is impossible. The target binding site (TBS) was designed to be located upstream of the sense strand of the T7 promoter. This design allows for the generation of a transcriptionally active double-stranded promoter through binding of the sensor DNA to the TBS and subsequent extension of the target DNA sequence. The construction of the sensor DNA is illustrated in Figure 1A.

[0124] Reporter genes were cloned into the pK7 plasmid after deletion of the T7 promoter using NdeI and SalI restriction enzymes (designated pK7 / dT7P-sfGFP, pK7 / dT7P-invertase, pK7 / dT7P-βgal, pK7 / dT7P-CDV, and pK7 / dT7P-NLuc, respectively). These genes (dT7P-sfGFP, dT7P-invertase, dT7P-βgal, dT7-CDV Ag, dT7P-NLuc) were amplified by PCR using a forward primer flanking the target-binding region and the (+) strand of the T7 promoter. The forward primer was synthesized using dUTP for the subsequent uracil-specific excision reaction (USER) to expose the (-) strand of the target-binding region and the T7 promoter. The primer sequences used are shown in the table below.

[0125]

[0126] Name sequence (5' to 3') [a] T7T-RCAAAAAACCCCTCAAGACCCGTTTAT715UP-FTCGATCCTGCGAAATTAATACGACTCACTATAGGGTGACCACAACGGTTTCCCTCTAGT715UP7U-FTCGAUCCUGCGAAATUAAUACGACUCACTAUAGGGUGACCACAACGGTTTCCCT CTAGYY-T715UP7U-F / 5'YakimaYellow / TCGAUCCUGCGAAATUAAUACGACUCACTAUAGGGUGACCACAACGGTTTCCCTCTAGT716UP8U-FTCGAUCCUGCGAAAUGUAAUACGACUCACTAUAGGGUGACCACAACGGTTTCCCTCTAG

[0127] [a] The underlined sequence represents the antisense strand of the T7 promoter.

[0128]

[0129] 160Nm of the PCR product was added to the USER mixture, incubated at 37°C for 2 hours, purified using a PCR clean-up kit (Promega), and used as sensor DNA.

[0130] Target DNA for TASER and IA-TASER (Invasive Amplification-TASER) assays was synthesized and purified by PAGE. To verify the detection of canine parvovirus type 2a (CPV2a, enBank accession number: NC_001539.1), a partial sequence of CPV2a was synthesized. To test the sequence specificity of the assay, a partial sequence of feline parvovirus (FPV) was also synthesized. The sequence information is as follows.

[0131] Name sequence (5' to 3') [a]Length (nt) CPV2a (4408C) CAAGAATTGTAACTTACTCAGATTTTTGGTGGAAAGGTAAATTAGTATTTAAAGCTAAACTAAGAGCCTCTCATACTTGGAATCCAATTCAACAAATGAGTATTAATGTAGATAACCAATTTAACTATGT130 FPV (4408A) CAAGAATTGTAACTTACTCAGATTTTTGGTGGAAAGGTAAATTAGTATTTAAAGCTAAACTAAGAGCATCTCATACTTGGAATCCAATTCAACAAATGAGTATTAATGTAGATAACCAATTTAACTATGT130 CPV / FPV-Invasive probe (CPV / FPV-Flap probe modified with FAM and BHQ) CTCATTTGTTGAATTGGATTCCAAGTATGAGAC33 CPV / FPV-Flap probe modified with FAM and BHQ BHQ) / 56-FAM / TCGATCCTGCGAAATGGCTC / iBHQ-1dT / TAGTTTAGCTTTAAATA38 CPV / FPV-Flap probe without modificationTCGATCCTGCGAAATGGCTCTTAGTTTAGCTTTAAATA38 CPV / FPV-Flap probe modified with phosphorothioate bondsTCGATCCTGCGAAATGGCTCTTAGTTTAGCTTT*A*A*A*T*A38 CPV / FPV-Flap probe modified with 2'MOETCGATCCTGCGAAATGGCTCTTAGTTTAGCTTTAAATA38 CPV / FPV-Flap probe modified with 2'MOE and phosphorothioate bonds (CPV / FPV-Flap probe modified with phosphorothioate bonds and2'MOE)TCGATCCTGCGAAATGGCTCTTAGTTTAGCTTT*A*A*A*T*A38CPV2aTTTGATTGTAAACCATGTAGACTAACACATACATGGCAAACAAATAGAGCATTGGGCTTACCACCATTTCTAAATTCTTTGCCTCAATCTGAAGGAGCTACTAACTTTGGTGATATAGGAGTTCAACAAGATAAAAGACGTGGTGTAACTCAAATGGGAAATACAAACTATATTACTGAAGCTACTATTATGAGACCAGC200CPV2a-Invasive probeATCTTGTTGAACTCCTATATCACCAAAGTTAGTAA35CPV2a-Flap probe modified with phosphorothioate bonds and 2'MOE)TCGATCCTGCGAAATGCTCCTTCAGATT*G*A*G*G*C33

[0132] [a] Underlined sequences indicate SNPs between CPV2a and FPV DNA. Italics indicate the sequence of the trigger DNA. * indicates a phosphorothioate bond. Bold indicates a 2'-O-methoxy-ethyl (2'MOE) modified base.

[0133]

[0134] TASER analysis

[0135] To an annealing solution (30 mM HEPES, pH 7.5, and 100 mM potassium acetate), 5 μL of target nucleic acid, sensor DNA (30 nM if encoding sfGFP; 10 nM if encoding NLuc, invertase, or β-gal; 2.5 nM if encoding CPV Ag), and nuclease-free water (up to 10 μL) were added. To allow the target nucleic acid to anneal to the TBS of the sensor DNA, the mixture was heated to 95 °C for 5 min and then cooled to room temperature. After annealing, 3 μL of the solution was discarded and mixed with 12 μL of a premix solution containing the following composition:

[0136] 57 mM HEPES-KOH (pH 7.5), 1.2 mM ATP, 0.85 mM each of CTP, GTP, and UTP, 2 mM DL-dithiothreitol, 0.17 mg / mL of E. coli total tRNA mixture (from strain MRE600), 0.64 mM cAMP, 90 mM potassium glutamate, 80 mM ammonium acetate, 12 mM magnesium acetate, 34 μg / mL folinic acid (l-5-formyl-5,6,7,8-tetrahydrofolic acid), 1.5 mM each of 20 amino acids, 67 mM creatine phosphate, 3.2 μg / mL creatine kinase, 0.2 U / μL E. coli DNA Polymerase I, 0.25 mM dNTPs, and 26% (v / v) extract from E. coli BL21Star(DE3) S12.

[0137] After incubating the mixture at 30°C for 0.5 h, an aliquot of the solution was collected to measure the activity of the cell-free synthesized protein.

[0138]

[0139] IA-TASER analysis

[0140] For IA-TASER analysis, the IA reaction was performed in a reaction mixture containing the following components:

[0141] 5 μl of target viral ssDNA, 1 μM flap probe (containing phosphorothioate linkages and 2'-O-methoxyethyl (2'MOE) base modifications at the last five 3'-terminal nucleotides), 100 nM each of invasive probes, sensor DNA (30 nM if encoding sfGFP; 10 nM if encoding NLuc, β-gal, or invertase; 2.5 nM if encoding CPV Ag), 1.6 U / μL FENI (Enzynomic, Cat# M025S, SEQ ID NO: 1), and nuclease-free water (up to 10 μl). The flap region of the flap probe has a T7UP(15 / 00) oligonucleotide complementary to the target binding region of the sensor DNA. The reaction mixture was incubated at 65°C for 2 h. After the IA reaction, 3 μL of the solution was discarded and mixed with 12 μL of the premix solution used in the TASER analysis.

[0142]

[0143] TASER signal analysis

[0144] In TASER or IA-TASER assays using sfGFP as the reporter, 10 μL of the completed assay reaction sample was mixed with 190 μL of PBS buffer and transferred to a 96-well microplate for fluorescence intensity measurement. In experiments using NLuc as the reporter enzyme, 10 μL of the completed assay reaction sample was diluted with 40 μL of PBS buffer and mixed with an equal volume of 2 X assay buffer. After 5 minutes at room temperature, the luminescence intensity was measured using a CLARIOStar microplate reader. Sensor DNA encoding β-gal was used for colorimetric measurement of nucleic acids. Ten μL of the assay sample was incubated with 90 μL of 300 μM chlorophenol red-β-D-galactopyranoside (CPRG) in a 96-well clear plate at room temperature for 30 minutes to develop a purple color, and the absorbance was measured at 575 nm using a spectrophotometer. In experiments using a personal glucose meter (PGM) as the measuring device, the TASER assay was performed using a sensor DNA encoding a bacterial invertase. A 15 μL sample of the completed assay reaction was mixed with an equal volume of 2 X sucrose (200 mM sucrose and 50 mM KH2PO4, pH 6.0) buffer. The mixed solution was incubated at 37 °C for 10 minutes and then briefly heated to 95 °C to terminate the hydrolysis reaction. The mixture was clarified by centrifugation at 13,000 × g for 5 min, and 5 μL of the supernatant was spotted onto a strip, and the glucose titer was measured using PGM (Accu-Check Inform II, Roche Diagnostics, Mannheim, Germany). For target nucleic acid detection using a Lateral Flow Immunoassay (LFIA) kit (BIONOTE, Hwaseong, Korea), a Rapid CDV Ag kit was used.In this case, the protein coding sequence of the DV antigen protein was used as the sensor DNA for TASER analysis. After incubation for 1 hour, 15 μL of the completed assay reaction was diluted with 45 μL of PBS buffer, and 50 μL of this was loaded into the LFIA kit. For statistical analysis of the results, the target band intensity on the strip was imaged and quantified using Image J. SPSS version 22.0 software (SPSS, Chicago, IL). Data with a p-value less than 0.005 were considered statistically significant (*p-value < 0.05, **p-value < 0.01, ***p-value < 0.005).

[0145]

[0146] Experimental results

[0147] To initiate sfGFP synthesis, we performed proof-of-concept tests for the TASER assay using the (+) strand of the T7 promoter as a model target DNA. The complete double-stranded sfGFP DNA sequence was obtained by PCR amplification of the pK7 / dT7-sfGFP plasmid using specific primers (T715UP-F and T7T-R). The forward primer featured a 15-nucleotide sequence upstream of the (+) strand of the T7 promoter. Furthermore, the forward primer was synthesized using dUTP (T715UP7U-F), and the resulting uracil-containing PCR product was treated with a uracil-specific cleavage reagent (USER) to produce sensor DNA exposing the (-) sequence of the T7 promoter (Figures 1A and 1B).

[0148] While PCR products generated using uracil-containing primers showed similar efficiencies in sfGFP production to those obtained with dTTP, sensor DNA obtained by USER treatment of uracil-containing primer PCR products was incapable of protein synthesis. This is because USER treatment renders the T7 promoter region single-stranded, and T7 RNA polymerase strictly requires a double-stranded T7 promoter. However, as shown in Figure 2C, cell-free synthesis of sfGFP by sensor DNA was successfully achieved when the exposed (-) T7 promoter sequence in the sensor DNA was annealed with the oligonucleotide T7UP(00 / 18), which contains the (+) strand of the T7 promoter. Sensor DNA annealed with oligonucleotide T7UP(00 / 18) produced sfGFP in approximately 48% yield compared to the control reaction using an intact double-stranded PCR product.

[0149] Additionally, higher levels of sfGFP production were achieved by progressively extending the 5' end of the T7 promoter (+) strand to form additional base pairs with the sensor DNA. Using the oligonucleotides listed in Table 3, sfGFP production increased proportionally with the number of additional base pairs upstream of the T7 promoter.

[0150]

[0151] Name Sequence (5' to 3') [a] Length (nucleotides) T7UP (00 / 18) [b]TAATACGACTCACTATAG18T7UP(02 / 18)ATTAATACGACTCACTATAG20T7UP(04 / 18)AAATTAATACGACTCACTATAG22T7UP(06 / 18)CGAAATTAATACGACTCACTAT AG24T7UP(04 / 13)AAATTAATACGACTCAC17T7UP(07 / 10)GCGAAATTAATACGACT17T7UP(13 / 04)GATCCTGCGAAATTAAT17T7UP(15 / 00)TCGATCCTGCGAAAT15

[0152] [a] The underlined T7 sequence represents the nucleotide that anneals to the (-) T7 promoter sequence of the sensor DNA. b] The number in parentheses represents the number of base pairs around the T7 promoter (+) strand of the sensor DNA. For example, (04 / 18) represents 4 nucleotide base pairs of the upstream target DNA and 18 nucleotide base pairs of the T7 promoter (+) strand.

[0153]

[0154] When six or more additional base pairs were formed between the target and sensor DNA, 80% of the yield of complete double-stranded DNA was reached (Fig. 2C). This improvement in production may be attributed to the stabilization of the target-probe complex due to the increased number of base pairs. When 50 nM of oligonucleotide T7UP(06 / 18) was used as the target DNA, the signal-to-background ratio (S / B) was 2.0 χ 10 2 From these results, it was confirmed that exogenously added oligonucleotides can effectively activate the expression of reporter proteins encoded by sensor DNA.

[0155]

[0156] Activation of transducers by various target sequences

[0157] Despite the promising findings of the above experiments, the T7 promoter strand is not readily usable as a target recognition component for the detection of nucleic acid sequences of interest. To realize the concept of the TASER assay, the TBS of the sensor DNA must be located separately upstream of the promoter sequence. However, because transcription by the T7 promoter requires at least partial duplexing, it was predicted that annealing the target to the TBS would not trigger transcription of the sensor DNA. This was further confirmed by in vitro transcription (IVT) experiments using purified T7 RNA polymerase. To determine the minimum length of double-stranded T7 promoter required for transcription, IVT experiments were performed in which oligonucleotides designed to bind to T7 promoter strands of various lengths were supplied to the sensor DNA.

[0158] As the double-stranded region moved progressively upstream from the T7 promoter sequence, transcript levels decreased significantly, with virtually no transcripts detected when the double-stranded region included fewer than 7 nucleotides to the 5'-end of the T7 promoter. Significant transcript levels were observed only when oligonucleotides annealed to 13 or more nucleotides of the T7 promoter sequence (Fig. 3).

[0159] However, in contrast to the IVT experiments above, duplex formation of the T7 promoter region was not required for reporter protein production during the TASER assay using the E. coli extract (S12 extract). When the sensor DNA was annealed with the same set of oligonucleotides used in the IVT experiments and transferred to the reaction mixture for the TASER assay, a significant amount of sfGFP was also produced from the sensor DNA annealed to the oligonucleotide that did not contain the T7 promoter sequence (T7UP(15 / 00), Figure 4A). Because the S12 extract used in the TASER assay contains most of the cellular enzymes, we speculated that the oligonucleotides annealed to the upstream sequence would be extended into the T7 promoter region by DNA polymerase I (Pol I) in the S12 extract, making the sensor DNA transcribable (Figure 4B). To verify this conjecture, sfGFP production was analyzed by TASER analysis by supplementing the S12 extract with purified Pol I (Fig. 5A) and dNTPs (Fig. 5B). Under optimized conditions (S12 extract supplemented with 0.2 U / μL Pol I and 0.25 mM dNTPs), the fluorescence intensity generated by the sensor DNA annealed to T7UP(15 / 00) reached approximately 180% of that in the reaction performed without Pol I and dNTPs (Fig. 4C). Consistent with this, the sensor DNA annealed to T7UP(15 / 00) was successfully transcribed in the IVT reaction when Pol I and dNTPs were supplemented (Fig. 4D).

[0160] We also used the PURExpress In Vitro Protein Synthesis Kit (PURE System, NEB) to confirm that Pol I-mediated elongation was responsible for successful expression of the sensor DNA. Unlike extract-based cell-free systems, the PURE system consists of purified components required for translation and thus does not require other cellular enzymes. Similar to the IVT reaction using purified T7 RNA polymerase, sensor DNA annealed to the oligonucleotide T7UP(15 / 00) did not produce sfGFP fluorescence in the PURE reaction mixture. However, when Pol I and dNTPs were supplemented, sfGFP synthesis was promoted in the PURE system at levels similar to the control reaction using intact double-stranded DNA (Fig. 4E).

[0161] In summary, the upstream sequence of the T7 promoter strand of the sensor DNA can be used as an effective TBS that can be flexibly programmed for TASER analysis of various target sequences.

[0162]

[0163] Flexibility of nucleic acid measurement proteins through cell-free transduction activity

[0164] Compared to conventional nucleic acid testing methods, the TASER assay offers greater flexibility in signal measurement methods. For example, when testing the detection of T7UP(15 / 00) DNA using sensor DNA encoding various reporter proteins, the TASER assay was able to detect the target nucleic acid based on various physical properties, such as color, luminescence, electrical signal, band intensity, and fluorescence (Figure 6). Detection sensitivity ranged from nanomolar (nM) to picomolar (pM) depending on the detection method used.

[0165] For example, a commercially available canine distemper virus (CDV) antigen test kit requires at least 5 nM of a trigger DNA encoding the nucleocapsid protein of CDV to detect a visible band on the strip when using a sensor DNA encoding the nucleocapsid protein of CDV, whereas the TASER assay can detect the same target DNA with only 5 Pm of the trigger DNA using the NLuc encoding sensor DNA (Figure 7).

[0166]

[0167] Introduction of the IA stage for sensitive detection of general-purpose targets.

[0168] In the previous examples, the preparation of sensor DNA involved several steps, including USER treatment of PCR products to expose single-stranded TBS and T7 promoters, and this procedure had to be repeated for each target nucleic acid sequence. Therefore, to overcome the complexity of sensor DNA preparation, IA technology was integrated into the TASER assay.

[0169] IA involves the use of an endonuclease, such as FENI, which recognizes and cleaves flap sequences at the junction between double-stranded and single-stranded DNA. Leveraging the unique activity of FENI, a trigger DNA was constructed to activate the TASER assay.

[0170] As illustrated in Figure 8, IA requires the adjacent binding of upstream and downstream oligonucleotides (referred to as invasion probes and flap probes, respectively) to the target nucleic acid to form a specific substrate structure for FENI to cleave the flap sequence. Using a flap probe with a common sequence that acts as a universal trigger DNA to activate the sensor DNA has the advantage of allowing the same TASER mixture to be used for different target nucleic acids without the need to redesign the sensor DNA. Furthermore, since TASER analysis is performed using an amplified amount of IA-producing trigger DNA, it is expected that combining IA and TASER reactions will improve the sensitivity of nucleic acid detection.

[0171] After confirming that the IA reaction successfully occurred when using a 6-carboxyfluorescein-labeled flap (Fig. 9), an IA-TASER assay was performed to detect canine parvovirus (CPV2a) using a chemically synthesized CPV2a DNA sequence (4341-4470, see Table 2) as a target nucleic acid. As expected, the IA reaction generated short-stranded DNA of the flap sequence that successfully initiated the TASER reaction (Fig. 10A). Furthermore, the specificity of the assay was confirmed using feline parvovirus (FPV), which differs from CPV2a by only one nucleotide in the conserved region (A4408C). In the IA-TASER assay, a fluorescent signal was detected only from the CPV2a target DNA that completely overlapped with the probe at the cleavage site, whereas no signal was detected from the FPV DNA (Fig. 10B).

[0172]

[0173] However, a background signal 25 times higher than that in the standard TASER assay was observed in the IA-TASER assay. This background signal was caused by the annealing of the intact flap probe to the sensor DNA. Since the 3'-terminal portion of the hybridized flap probe was removed by the exonuclease activity of Pol I, allowing the target binding sequence to form a complete duplex with TBS to be extended, the reporter protein could be synthesized even in the absence of the target sequence (Fig. 11B). In addition, it was confirmed that combining a phosphorothioate linkage and a 2'MOE modification could effectively eliminate the background signal by preventing the degradation of the 3'-terminus of the 3' flap sequence without affecting the binding or cleavage of the flap probe by a structure-specific endonuclease (Fig. 11C). Next, the IA-TASER assay conditions were optimized to effectively generate trigger DNA from target DNA, including endonuclease concentration, reaction temperature, probe concentration, and reaction time (Figs. 12 and 13). The fluorescence signal generated by sfGFP increased continuously over time in the presence of 50 pM target DNA, but a 2-hour IA reaction time was determined to be optimal based on S / B. Finally, when a luminescence spectrometer was used as the measuring device for the NLuc-encoded sensor DNA, the IA-TASER assay was capable of detecting femtomolar levels of target nucleic acids. In contrast, assays using sfGFP and invertase as reporter proteins exhibited limits of detection in the picomolar range (Fig. 14). Figure 15 shows a strong linear relationship between the logarithms of target DNA concentrations of 1 pM to 1 Fm and 10 pM to 100 fM and the reporter protein signals measured by luminescence and fluorescence. The LODs were determined at 1.6 fM and 2.0 pM, respectively.

[0174]

[0175] Example 2. Detection of target nucleic acid using IA-TASER

[0176] Materials and Methods

[0177] The sensor DNA was designed to be activated to initiate synthesis of the encoded protein by annealing the trigger DNA to the trigger binding site (TBS) located upstream of the single-stranded T7 promoter as described in Example 1 above. Tth DNA polymerase (SEQ ID NO: 2) was purchased from ELPIS Biotech (Daejeon, Korea).

[0178]

[0179] Target RNA preparation

[0180] Pathogenic target RNAs were prepared by in vitro transcription (IVT). A chemically synthesized double-stranded DNA fragment encoding the 16S rRNA of BWA under the T7 promoter (Table 4) was cloned into the pTOP vector using the TOPcloner PCR Cloning Kit (Enzynomics, Korea). PCR amplification was performed using a forward primer for the T7 promoter and a reverse primer targeting the 3'-terminal sequence of each 16S rDNA sequence. The amplified PCR products were purified using a PCR clean-up kit and transcription was performed using the RiboMAX Large-Scale RNA Production System-T7 (Promega). After the IVT reaction, the transcripts were treated with DNase 1 (1 μg / 1 Unit) at 37°C for 30 minutes and then purified using the RNeasy mini Kit (Qiagen). The transcript concentration was measured at 280 nm using a UV-Vis spectrophotometer.

[0181] Primer information used: NameSequence (5' to 3') [a]T716UP8U-FTCGAUCCUGCGAAUGUAAUACGACUCACTAUAGGGUGACCACAACGGTTTCCCTCTAGT7T-RCAAAAAACCCCTCAAGACCCGTTTAE.coli-V4_InvasiveGACTCAAGCTTGCCAGTATCAAE.coli-V4_Flap wF / Q / 56-FAM / TCGATCCTGCGAAATGATGCAG / iBHQ-1dT / TCCCAGGTTT7PTAATACGACTCACTATAGGB. anthracis-RCACCCCAATCATCTGTCCCAF. tularensis-RCACCCCAGTCATGAATCACTY. pestis-RCACCCCAGTCATGAATCACAAAAGTB. pseudomallei-RCACCCCAGTCATGAATCCTACCB. abortus-RCACCCCAGTCGCTGA

[0182] IVT 주형을 위한 타겟 RNA를 코딩하는 DNA 주형 서열NameSequence (5' to 3') [a]E. coliH157:O7_16sV4TAATACGACTCACTATAGGGCGCACGCAGGCGGTTTGTTAAGTCAGATGTGAAATCCCCGGGCTCAACCTGGGAACTGCATCTGATACTGGCAAGCTTGAGTCTCGTAGAGGGGGGTAGAAE. coliH157:O7TAATACGACTCACTATAGTTGGTGCCTTCGGGAACTGTGAGACAGGTGCTGCATGGCTGTCGTCAGCTCGTGTTGTGAAATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTATCTTTTGTTGCCAGCGGTCCGGCCGGGAACTCAAAGGAGACTGCCAGTGATAAACTGGAGGAAGGTGGGGATGACGTCAAGTCATCATGGCCCTTACGACCAGGGCTACACACGTGCTACAATGGCGCATACAAAGAGAAGCGACCTCGCGAGAGCAAGCGGACCTCATAAAGTGCGTCGTAGTCCGGATTGGAGTCTGCAACTCGACTCCATGAAGTCGGAATCGCTAGTAATCGTGGATCAGAATGCCACGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCATGGGAGTGGGTTGCAAAAGAAGTAGGTAGCTTAACCTTCGGGAGGGCGCTTACCACTTTGTGATTCATGACTGGGGTGB.anthracisTAATACGACTCACTATAGAGAGTGACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTGATCTTAGTTGCCATCATTAAGTTGGGCACTCTAAGGTGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAATCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGGACGGTACAAAGAGCTGCAAGACCGCGAGGTGGAGCTAATCTCATAAAACCGTTCTCAGTTCGGATTGTAGGCTGCAACTCGCCTACATGAAGCTGGAATCGCTAGTAATCGCGGATCAGCATGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCACGAGAGTTTGTAACACCCGAAGTCGGTGGGGTAACCTTTTTGGAGCCAGCCGCCTAAGGTGGGACAGATGATTGGGGTGF. tularensisTAATACGACTCACTATAGGGCTGTCGTCAGCTCGTGTTGTGAAATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCCTATTGATAGTTACCATCATTAAGTTGGGTACTCTATTGAGACTGCCGCTGACAAGGCGGAGGAAGGTGGGGACGACGTCAAGTCATCATGGCCCTTACGACCAGGGCTACACACGTGCTACAATGGGTATTACAGAGGGCTGCGAAGGTGCGAGCTGGAGCGAAACTCAAAAAGGTACTCTTAGTCCGGATTGCAGTCTGCAACTCGACTGCATGAAGTCGGAATCGCTAGTAATCGCAGGTCAGAATACTGCGGTGAATACGTTCCCGGGTCTTGTACACACCGCCCGTCACACCATGGGAGTGGGTTGCTCCAGAAGTAGATAGCTTAACGAATGGGCGTTTACCACGGAGTGATTCATGACTGGGGTGY.pestisTAATACGACTCACTATAGGTGCTGCATGGCTGTCGTCAGCTCGTGTTGTGAAATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTATCCTTTGTTGCCAGCACGTAATGGTGGGAACTCAAGGGAGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAGTCATCATGGCCCTTACGAGTAGGGCTACACACGTGCTACAATGGCAGATACAAAGTGAAGCGAACTCGCGAGAGCCAGCGGACCACATAAAGTCTGTCGTAGTCCGGATTGGAGTCTGCAACTCGACTCCATGAAGTCGGAATCGCTAGTAATCGTAGATCAGAATGCTACGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCATGGGAGTGGGTTGCAAAAGAAGTAGGTAGCTTAACCTTCGGGAGGGCGCTTACCACTTTGTGATTCATGACTGGGGTGB. pseudomalleiTAATACGACTCACTATAGGCATGGCTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTGTCCTTAGTTGCTACGCAAGAGCACTCTAAGGAGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAGTCCTCATGGCCCTTATGGGTAGGGCTTCACACGTCATACAATGGTCGGAACAGAGGGTCGCCAACCCGCGAGGGGGAGCCAATCCCAGAAAACCGATCGTAGTCCGGATTGCACTCTGCAACTCGAGTGCATGAAGCTGGAATCGCTAGTAATCGCGGATCAGCATGCCGCGGTGAATACGTTCCCGGGTCTTGTACACACCGCCCGTCACACCATGGGAGTGGGTTTTACCAGAAGTGGCTAGTCTAACCGCAAGGAGGACGGTCACCACGGTAGGATTCATGACTGGGGTGB.abortusTAATACGACTCACTATAGGAGCGCAACCCTCGCCCTTAGTTGCCAGCATTCAGTTGGGCACTCTAAGGGGACTCTAAGGTGATAAGCCGAGAGGAAGGTGGGGATGACGTCAAGTCCTCATGGCCCTTACGGGCTGGGCTACACACGTGCTACAATGGTGGTGACAGTGGGCAGCGAGCACGCGAGTGTGAGCTAATCTC CAAAAGCCATCTCAGTTCGGATTGCACTCTGCAACTCGAGTGCATGAAGTTGGAATCGCTAGTAATCGCGGATCAGCATGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCATGGGAGTTGGTTTTACCCGAAGGCGCTGTGCTAACCGCAAGGAGGCAGGCGACCACGGTAGGGTCAGCGACTGGGGTG.

[0183] [a] Underlined letters indicate the T7 promoter sequence.

[0184]

[0185] IA analysis

[0186] Target ssDNA and RNA for IA were chemically synthesized or prepared by in vitro transcription, respectively. IA reactions were performed in nuclease-free water (up to 10 μL) using 1 μM target nucleic acid, 1 μM flap probe with internal modifications of fluorescence (FAM) and quencher (BHQ1), 100 nM each of invasive probes, and 0.25 U / μL Tth DNA polymerase or 1.6 U / μL FEN1 (SEQ ID NO: 1). Gel electrophoresis analysis was performed using a 16% denaturing (8 M UREA) polyacrylamide gel. FRET analysis was performed using a real-time PCR system (CFX Opus 96, Biorad) to measure the real-time fluorescence emitted from the cleaved flap probe.

[0187]

[0188] IA-TASER analysis

[0189] Similar to Example 1 above, the reaction was performed in a mixture containing 7.5 μL of target RNA, 1 μM flap probe with phosphorothioate linkages and 2'-O-methoxyethyl (2'MOE) base modification at the 5 nucleotides at the 3' end, 100 nM invasion probes each, 2.5 U / μL Tth DNA polymerase, and nuclease-free water (up to 15 μL). The reaction was incubated at 60°C for 1 h, and 7.5 μL of the completed IA reaction mixture was discarded and mixed with an equal volume of 10 nM sensor DNA in annealing buffer (10 mM HEPES, pH 7.5, and 100 mM potassium acetate). The mixture was heated at 95°C for 5 min to allow the trigger DNA generated during the IA reaction to anneal to the TBS of the sensor DNA, and then cooled to room temperature.

[0190] For TASER analysis, 3 μL solution from this mixture was discarded and mixed with 12 μL of TASER assay solution containing the following components: 57 mM HEPES-KOH (pH 7.5), 1.2 mM ATP, 0.85 mM each of CTP, GTP and UTP, 2 mM DL-dithiothreitol, 0.17 mg / mLE. E. coli total tRNA mixture (MRE600 strain), 0.64 mM cAMP, 90 mM potassium glutamate, 80 mM ammonium acetate, 12 mM magnesium acetate, 34 μg / mL folic acid (1-5-formyl-5,6,7,8-tetrahydrofolic acid), 1.5 mM each of 20 amino acids, 67 mM creatine phosphate, 3.2 μg / mL creatine kinase, 0.2 U / μL E. coli DNA polymerase I, 0.25 mM dNTPs, and 26% (v / v) S12 extract prepared from E. coli strain BL21Star (DE3) (Park and Kim, 2021).

[0191]

[0192] Multiplex detection of 16S rRNA in BWA using IA-TASER analysis

[0193] E. coli O157:H7 was mixed with five potential BWAs in vitro: Bacillus anthracis (B. anthracis), Francisella tularensis (F. tularensis), Yersinia pestis (Y. pestis), Burkholderia pesudomallei (B. pseudomallei), and Brucella abortus (B. abortus) (Dennis et al. 2001; Inglesby et al. 2000; Inglesby et al. 1999; Pappas et al. 2006; Wiersinga et al. 2018), and the concentration of each rRNA was adjusted to 100 nM. IA-TASER analysis was performed on the mixture using flap probes and invasive probes specific for each target 16S rRNA. In experiments simulating 16S rRNA in biological matrices, 10 μg / mL of 16S rRNA mixture was added. 16S rRNA in this mixture was purified using the RNeasy Mini Kit (Qiagen) and then analyzed by IA-TASER analysis.

[0194]

[0195] Probe sequences used

[0196] The IA probe oligonucleotide sequences are shown in Table 6 below.

[0197] TargetpathogenRegion in 16S rRNAFlap probe [a]Invasive probeE. coliH157:O7660CTCGATCCTGCGAAATGAGACTCAAGC*T*T*G*C*CGAATTCTACCCCCCTCTACA637CTCGATCCTGCGAAATGATGCAGTTCCC*A*G*G*T*TGACTCAAGCTTGCCAGTATCAA1006CTCGATCCTGCGAAATGTTCTGTGGATGTC*A*A*G*A*CCACCAATCCATCTCTGGAAAA1120TTCGATCCTGCGAAAGATAAGGGTTG*C*G*C*T*CGACCGCTGGCAACAAATB. anthracis195CTCGATCCTGCGAAATGAAGCCGCCTTTCAATT*T*C*G*A*AGTCCATCCATAAGTGACAGCCA477CTCGATCCTGCGAAATGTTAGGTACCGTCAA*G*G*T*G*CGTAGTTAGCCGTGGCTTTCTGA576CTCGATCCTGCGAAATGAAACCACC*T*G*C*G*CGGGCTTTCACATCAGACTTAAA1020ATCGATCCTGCGAAATCCCGAAGGAGAAGC*C*C*T*A*TCACCACCTGTCACTCTGCA1308CTCGATCCTGCGAAATGCGAGTTGCAGCCTAC*A*A*T*C*CTAGCGATTCCAGCTTCATGTAGAF.tularensis119CTCGATCCTGCGAAATGTATCCCCCTCAAATGGA*C*A*G*A*TCGGTATTAACAGTCGTTTCCAACTGA347CTCGATCCTGCGAAATGTTGCCCCCATTGTCCAA*T*A*T*T*CATGGCATTGCTGGATCAGGA419CTCGATCCTGCGAAATGGCTTTCCTCCCCAAC*T*A*A*A*GCCCCAAGGCTATTAACCTTGAA542CTCGATCCTGCGAAATGACCCTTTAC*G*C*C*C*ACTGACTTAACAAACCACCTACAA816CTCGATCCTGCGAAATGAGCCTTTACACCGAC*T*C*C*A*ACGTTAGCTGCGCCACTAA1022CTCGATCCTGCGAAATGTGCAGCACCTGT*C*A*C*T*GCGAGCTGACGACAGCCA1087ATCGATCCTGCGAAATCAATAGGGGTTG*C*G*C*T*CGAGTACCCAACTTAATGATGGTAACTAA1236CTCGATCCTGCGAAATGCTCGCACCT*T*C*G*C*AACCTTTTTGAGTTTCGCTCCAA1259CTCGATCCTGCGAAATGTACCTTTTTGAGTTTCG*C*T*C*C*A AGACTGCAATCCGGACTAAGAA1330ATCGATCCTGCGAAATATTCTGACCTGCGATTA*C*T*A*G*CGGGAACGTATTCACCGCAGAY.pestis288ATCGATCCTGCGAAATACTAGCTAATCCCA*T*C*T*G*GTGAGCCATTACCCCACCA448CTCGATCCTGCGAAATGAGCGTATTAAACTC*A*A*C*C*CCTGCGAGTAACGTCAATGATTA450ATCGATCCTGCGAAATTGAGCGTATTAAACT*C*A*A*C*CCTGCGAGTAACGTCAATGAA591CTCGATCCTGCGAAATGCGGGGATTTC*A*C*A*T*CGCAGTTCCCACGTTAAGCA1220ATCGATCCTGCGAAATGCCATTGTAGC*A*C*G*T*GGAGTTCGCTTCACTTTGTATCA1250CTCGATCCTGCGAAATGGCTCTCGCG*A*G*T*T*CGACTTTATGTGGTCCGCTAB. pseudomallei273CTCGATCCTGCGAAATGTCCTCTCAGACCAG*C*T*A*C*TCCCAGTGTGGCTGGTCA413CTCGATCCTGCGAAATGGACAAAAGTGCTTTACA*A*C*C*C*GGGGTATTAGCCAGAATGATTTCTTTCCA562CTCGATCCTGCGAAATGCAAACCGC*C*T*G*C*GGGGGATTTCACATCGGTCTTAA569CTCGATCCTGCGAAATGGTCTTAGCAA*A*C*C*G*CCCCGGGGATTTCACATCA1103CTCGATCCTGCGAAATGCGTAGCAACTAAGGA*C*A*A*G*GGCAGTCTCCTTAGAGTGCTCTTA1197CTCGATCCTGCGAAATGACGTGTGAAGC*C*CT*A*C*CCCTCTGTTCCGACCATTGTATA1224CTCGATCCTGCGAAATGGCGACCCTCT*G*T*T*C*CCCCCTCGCGGGTTA1428CTCGATCCTGCGAAATGACCGTCCTCC*T*T*G*C*GAGTCATGAATCCTACCGTGGTAB.abortus171CTCGATCCTGCGAAATGATCATTTGCCGATAAATCTT*T*C*C*C*CAATCCAACGCGGGCCA383CTCGATCCTGCGAAATGTGAAAGAGCTTTACAACC*C*T*A*G *GGGTTACCGTCATTATCTTCACCGA506CTCGATCCTGCGAAATGTCCGCCTAC*G*T*G*C*GGGGATTTCACCCCTGACTTAAAAA551CTCGATCCTGCGAAATGGCAGTTCCG*G *G*G*T*TCCATACTCAAGACTTCCAGTATCAAAA747CTCGATCCTGCGAAATGGCTAACATTCATCGTTT*A*C*G*G*CCGAAGTGTAAACACCCCGACA889CTCGATCCTGCGA AATGCGCGTTGCTTCGAAT*T*A*A*A*CTCAAGGGCTGGTAAGGTTCTA1204CTCGATCCTGCGAAATGGAGATTAGCTCACA*C*T*C*G*CCAATCCGAACTGAGATGGCTTTTA.

[0198] [a] * indicates phosphorothioate bond, bold indicates 2'MOE bond.

[0199]

[0200] result

[0201] RNA detection

[0202] To address the issue of reduced FEN1-mediated cleavage of flap probes when hybridized to RNA (Fig. 18A), we employed Thermus thermophilus (Tth) DNA polymerase, which has been shown to excise flap sequences from RNA hybrid structures (Eis et al. 2001). When the same probe set was used, the FRET signal indicating flap sequence cleavage from RNA-hybridized flap probes was enhanced approximately 3.5-fold when mediated by Tth DNA polymerase compared to FEN1 (Fig. 18B).

[0203] Based on these results, Tth DNA polymerase was applied to generate trigger DNA in TASER analysis using RNA as a target sequence.

[0204]

[0205] Detection of microbial 16S rRNA using IA-TASER analysis

[0206] Sensor DNA encoding sfGFP and various concentrations of E. coli O157:H7 16S rRNA were used in the IA-TASER assay. As shown in Figure 19A, the limit of detection (LOD) was approximately 2 nM. When the sensor DNA was changed to DNA encoding NLuc, the detection sensitivity was significantly improved, achieving an LOD of 2.8 pM (Figure 19B).

[0207]

[0208] Multiple detection of 16S rRNA in BWA

[0209] After validation using E. coli O157:H7 rRNA, the analysis was extended to include five potential biological warfare agents (BWAs) (Bacillus anthracis (B. anthracis), Francisella tularensis (F. tularensis), Yersinia pestis (Y. pestis), Burkholderia pesudomallei (B. pseudomallei), and Brucella abortus (B. abortus)) to detect a broader spectrum of pathogenic 16S rRNA.

[0210] The probe sets in Table 6 were tested to analyze the 16S rRNA of six pathogenic strains. As shown in Figure 20A, significant signals were primarily observed when a specific 16S rRNA was matched with the appropriate probe set, resulting in a distinct diagonal pattern in the heat map. Conversely, when the 16S rRNA was matched with an uncorrelated probe, the generated signal was minimal and virtually indistinguishable from the background signal seen in analyses performed without the 16S rRNA species. Additionally, the specific detection ability of the TASER for the selective target 16S rRNA was tested in a scrambled mixture with unrelated RNA sequences.

[0211] To this end, six types of 16S rRNA were mixed at the same concentration (100 nM) and individual probe sets targeting the designated pathogens were provided, and then analyzed by IA-TASER analysis. As shown in Figure 21A, IA-TASER analysis properly identified the target 16S rRNA with S / B values ​​higher than 6.0 for all targets, while only background signal levels were observed when the target 16S rRNA was excluded (Figure 21B). This confirmed that IA-TASER analysis could clearly distinguish the target 16S rRNA from 16S rRNA of other strains.

[0212]

[0213] From the above description, those skilled in the art will understand that the present invention can be implemented in other specific forms without altering its technical spirit or essential characteristics. In this regard, it should be understood that the embodiments described above are illustrative in all respects and not restrictive. The scope of the present invention should be interpreted as encompassing all changes or modifications derived from the meaning and scope of the following claims and their equivalent concepts, rather than the detailed description above.

Claims

1. A composition for detecting a target nucleic acid comprising (i) sensor DNA, (ii) a flap probe, and (iii) FEN (flap endonuclease), The above (i) sensor DNA Trigger nucleic acid sequence binding region - promoter - reporter gene Contains the structure of, The above trigger nucleic acid sequence binding region and promoter are single-stranded DNA, and the reporter gene is double-stranded DNA. The above (ii) flap probe i) a trigger nucleic acid sequence and ii) a sequence capable of annealing to a target nucleic acid sequence, A composition characterized in that, when the target nucleic acid sequence is present, the ii) sequence capable of annealing to the target nucleic acid sequence is annealed to the target nucleic acid sequence, and the probe sequence and the target nucleic acid sequence form a non-complementary flap structure, so that the i) trigger nucleic acid sequence is cleaved by FEN (Flap endonuclease).

2. In paragraph 1, the flap probe (ii) 5' - (trigger nucleic acid sequence) - (sequence capable of annealing to target nucleic acid sequence) - 3' It has the structure of A composition comprising a modification at the 3' end that inhibits flap degradation.

3. A composition according to claim 1, wherein the target nucleic acid sequence is DNA or RNA.

4. A composition according to claim 1, wherein the reporter gene encodes a protein selected from the group consisting of a fluorescent protein, luciferase, galactosidase, and invertase; or an aptamer.

5. A composition according to claim 1, wherein the promoter is a T7 promoter.

6. A composition according to claim 1, wherein the composition further comprises a component necessary for performing at least one of cell-free replication, transcription, and synthesis.

7. A composition according to claim 6, wherein the composition comprises DNA polymerase and nucleotides.

8. A composition according to claim 7, wherein the DNA polymerase is DNA polymerase I (Pol I).

9. A composition according to claim 7, wherein the nucleotide is a dNTP.

10. A composition according to claim 1, wherein the composition further comprises a crude extract of E. coli.

11. A step of adding a sample to the composition of any one of clauses 1 to 10; and A method for detecting a target nucleic acid, comprising the step of measuring the expression level of a reporter gene by performing at least one reaction among cell-free replication, transcription and protein synthesis.

12. A method according to claim 11, wherein the sample is separated from a living organism.

13. A method according to claim 11, wherein the target nucleic acid is DNA of a virus.

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