Method for detecting target molecules based on cell-free protein synthesis and retroreflection signal

Abstract

The present invention relates to a sensor DNA that can be used in a method for easily detecting a target nucleic acid with high sensitivity and, more specifically, to: a sensor DNA for detecting a target nucleic acid, comprising i) a sequence complementary to the target nucleic acid or a trigger nucleic acid, ii) a promoter, and iii) a sequence encoding a reporter protein comprising a first domain that specifically binds to a first peptide attached to a retroreflective particle and a second domain that specifically binds to a second peptide immobilized on a substrate; a kit comprising the sensor DNA; and a method for detection using same, wherein the method for detecting a target nucleic acid by using the sensor DNA according to the present invention exhibits high detection performance at a level of several femtomoles and can detect a target nucleic acid with just a simple optical detector such as a camera, thereby enabling easier detection of a target nucleic acid compared to conventional methods for detecting a target nucleic acid based on fluorescence, bioluminescence, and electrochemical signals. In addition, the method for detecting a target nucleic acid according to the present invention has an advantage in that a target nucleic acid to be detected can be easily changed by changing the sequence complementary to the target nucleic acid of the sensor DNA or by changing a probe pair for obtaining the trigger nucleic acid from the target nucleic acid.

Description

A method for detecting target substances based on cell-free protein synthesis and retroreflective signals

[0001] The present invention relates to a sensor DNA for detecting a target nucleic acid that can be used in a cell-free protein synthesis capable of easily detecting a target nucleic acid with high sensitivity and a method for detecting a target nucleic acid based on a retroreflective signal, a kit including the same, and a method for detecting a target nucleic acid using the same.

[0002]

[0003] Target nucleic acid detection technology has potential applications in a wide range of fields, including food analysis, forensics, genetics, and molecular diagnostics. A representative target nucleic acid detection technology is PCR, which amplifies DNA in a short period of time (Saiki, R., et al. Science 239, 487-91, 1998). These target nucleic acid detection technologies are continuously evolving to improve detection sensitivity and convenience. In particular, in food analysis, rapid and effective detection of foodborne pathogens in food samples is crucial for controlling foodborne pathogens. Conventional methods based on microbial growth are used to detect foodborne pathogens in the food industry, but these methods are time-consuming (Gracias, KS, et al., Can J Microbiol 2004, 50 (11), 883-90; Kim, TH, et al., Anal Chem 2014, 86 (8), 3841-85-6). To replace these, methods based on nucleic acid amplification technology and enzyme-linked immunosorbent assays have been developed for pathogen detection. However, these methods require specialized equipment and skilled technicians for sample processing and signal measurement, making their widespread application to food contamination management difficult (Law, JW, et al., Front Microbiol 2014, 5, 770). Therefore, a detection method that enables simple on-site sample processing and signal measurement is needed.

[0004] On one hand, cell-free protein synthesis systems (CFPS) consist of molecular machinery for transcription and translation under customized and optimized biochemical conditions, and can produce recombinant proteins of interest from genes without living cells (Carlson, ED, et al., Biotechnol Adv 2012, 30 (5), 1185-94.; Silverman, AD, et al., Nat Rev Genet 2020, 21 (3), 151-170.; Garenne, D., et al., Cell-free gene expression. Nature Reviews Methods Primers 2021, 1 (1)). When utilizing cell-free protein synthesis, transcription and translation are performed in the presence of a target substance, so the signal is amplified, allowing for high sensitivity detection of the target substance. In addition, since there is no cell culture step, reporter proteins can be synthesized from the target substance simply and quickly. Previously, a method was reported in which reporter proteins designed to generate fluorescent, bioluminescent, or electrochemical signals were synthesized and measured using spectroscopic or amperometric detectors (Zhang, L. et al., Biotechnol J 2020, 15 (9), e2000187.), but the detection of fluorescent, bioluminescent, or electrochemical signals requires specialized equipment and techniques.

[0005] On the other hand, retroreflection refers to the phenomenon in which a significant portion of directed light is returned to the light source, and the observation of retroreflected light has the advantage of being performed with equipment consisting of a light source and a simple optical detector, so retroreflective signals can be considered as an alternative method for detection (Han, YD, et al., Biosens Bioelectron 2022, 207, 114202.). Retroreflectors, which are materials that cause retroreflection, have proven useful in applications such as road safety, signage, and photography, and it has been reported that retroreflective Janus microparticles (RJPs) can be used as optical probes (Han, YD, et al., ACS Appl Mater Interfaces 2016, 8 (17), 10767-74.). Janus microparticles are spheres made of silica with half of the structure coated with a metal layer, and light entering through the uncoated surface is reflected toward the light source. Although detection methods for proteins and nucleic acids using the above Janus microparticles have been reported (Kim, KR, et al., Lab Chip 2019, 19 (23), 3931-3942.; Chun, HJ, et al., ACS Sens 2018, 3 (11), 2261-2268.), directional alignment is required to use retroreflective particles as optical probes, making it difficult to be compatible with detection methods based on cell-free synthetic systems.

[0006] From this point of view, the present inventors have made great efforts to develop a detection technology that has both the advantage of a cell-free synthesis system that can detect a target substance in a short period of time with high detection sensitivity and the advantage of a retroreflective signal that can be easily measured using only a simple optical detector such as a camera. As a result, they have designed a reporter protein including a first domain that specifically binds to a retroreflective particle and a second domain that specifically binds to a substrate, and designed a sensor DNA that cell-free synthesizes the reporter protein when a target nucleic acid is present, thereby confirming that a target nucleic acid can be detected with high sensitivity and specificity using only a simple optical detector, and have completed the present invention.

[0007] The above information described in this background section is solely intended to enhance understanding of the background of the present invention and may not include information that constitutes prior art already known to a person of ordinary skill in the art to which the present invention pertains.

[0008]

[0009] Summary of the invention

[0010] An object of the present invention is to provide a sensor DNA for target nucleic acid detection based on cell-free synthesis and retroreflective signal.

[0011] The purpose of the present invention is to provide a kit for detecting a target nucleic acid comprising the sensor DNA.

[0012] The purpose of the present invention is to provide a method for detecting a target nucleic acid using the sensor DNA.

[0013]

[0014] In order to achieve the above purpose, the present invention provides a sensor DNA for detecting a target nucleic acid, comprising: i) a sequence complementary to a target nucleic acid or a trigger nucleic acid; ii) a promoter; and iii) a sequence encoding a reporter protein.

[0015] A sensor DNA for detecting a target nucleic acid is provided, wherein the sequence and promoter complementary to the target nucleic acid or trigger nucleic acid are single-stranded, the sequence encoding the reporter protein is double-stranded or single-stranded, the reporter protein comprises a first domain and a second domain, the first domain specifically binds to a first peptide attached to a retroreflective particle, and the second domain specifically binds to a second peptide immobilized on a substrate.

[0016] The present invention also provides a kit for detecting a target nucleic acid comprising the sensor DNA.

[0017] The present invention also provides a method for detecting a target nucleic acid, comprising the following steps:

[0018] (a) a step of mixing a sample containing a target nucleic acid with the sensor DNA, hybridizing the target nucleic acid and the sensor DNA, and then treating the sample with a DNA polymerase to form a double-stranded nucleic acid;

[0019] (b) a step of synthesizing a reporter protein from the formed double-stranded nucleic acid;

[0020] (c) mixing the synthesized reporter protein with the retroreflective particles to which the first peptide is attached, and treating the substrate to which the second peptide is fixed, thereby arranging the retroreflective particles in a certain direction on the substrate; and

[0021] (d) A step of detecting a retroreflective signal generated from retroreflective particles by irradiating light.

[0022] The present invention also provides a method for detecting a target nucleic acid, comprising the following steps:

[0023] (a) a step of hybridizing a trigger nucleic acid obtained from a sample containing a target nucleic acid with the sensor DNA containing a sequence complementary to the trigger nucleic acid, and then treating the hybrid with a DNA polymerase to form a double-stranded nucleic acid;

[0024] (b) a step of synthesizing a reporter protein from the formed double-stranded nucleic acid;

[0025] (c) mixing the synthesized reporter protein with the retroreflective particles to which the first peptide is attached, and treating the substrate to which the second peptide is fixed, thereby arranging the retroreflective particles in a certain direction on the substrate; and

[0026] (d) A step of detecting a retroreflective signal generated from retroreflective particles by irradiating light.

[0027]

[0028] Figure 1 illustrates the concept of a method for detecting a target single-stranded nucleic acid. A cell-free protein synthesis reaction converts the target nucleic acid into the expression of a reporter protein. This reporter protein then induces the orientation of retroreflective Janus particles on a substrate surface. The retroreflective signal is measured using a simple device consisting of a white light source and a camera. The image below is an example obtained using the proposed method.

[0029] Figure 2 shows a schematic representation of two potential methods for linking MDM2 and SpyCatcher003 via a peptide linker. Figure 2 (A) shows the structure of the MDM2-SpyCatcher003 protein, and (B) shows the structure of the SpyCatcher003-MDM2 protein. The protein structures of 3LNZ and 4MLI were used to represent each protein domain (MDM2 (pink), SpyCatcher003 (green)).

[0030] Figure 3 illustrates the characterization of reporter proteins. Figure 3 (A) shows the expression of MDM2-SpyCatcher003, SpyCatcher003-MDM2, and MDM2-MDM2-SpyCatcher003 using a cell-free protein expression system. The C-terminal His6 tag was detected using an anti-His6-horseradish peroxidase conjugate. (B) shows the results of electrophoresis confirming the binding between MDM2-SpyCatcher003 and the SpyTag003-Z domain. (C) shows the results of microscale thermophoresis analyzing the binding affinity between MDM2-SpyCatcher003 and PMI (N8A).

[0031] Figure 4 shows the binding affinity of MDM2-SpyCatcher003 and PMI (N8A) in a cell-free protein expression solution based on microscale thermophoresis (MST).

[0032] Figure 5 illustrates the procedure for immobilizing the first peptide and the second peptide (SpyTag003 and PMI(N8A)) onto the surface of a solid.

[0033] Figure 6 illustrates the assembly of retroreflective particles on a substrate surface via MDM2-SpyCatcher003. Protein structures of 3LNZ and 4MLI were used to represent MDM2-PMI(N8A) and SpyCatcher003-SpyTag003, respectively. MDM2-SpyCatcher003 is shown in green, PMI(N8A) in pink, and SpyTag003 in yellow. Azidolysine (*) was introduced at the N-terminus of PMI(N8A) and the C-terminus of SpyTag003, respectively.

[0034] Figure 7 shows the results of confirming the interaction between a retroreflective particle (A) to which a fluorescently labeled reporter protein (MDM2-SpyCatcher-GFP) and a first peptide (SpyTag003) are attached, and a substrate surface (B) to which a second peptide (PMI (N8A)) is immobilized. In Figure 7A, it can be confirmed that the reporter protein is attached to a portion of the surface of the retroreflective particle through the green fluorescence signal generated from GFP, and in Figure 7B, it can be confirmed that the reporter protein is immobilized on the substrate through the green fluorescence signal generated from GFP on the surface of the substrate to which PMI (N8A) is immobilized.

[0035] Figure 8 shows the results of retroreflective signal detection according to the concentration of purified MDM2-MDM2-SpyCatcher003 based on a substrate combined with retroreflective particles and PMI (N8A) combined with SpyTag003.

[0036] Figure 9 shows the detection performance of a combined technique of a retroreflective signal detection method and a cell-free protein synthesis (CFPS) reaction. Figure 9 (A) shows the structure of the gene construct used in the CFPS reaction. Figure 9 (B) shows the relationship between the log concentration of the DNA fragment indicated in (A) and the retroreflective signal. The ΔRJP number is the number of retroreflective particles in each sample minus the number of retroreflective particles in the control group. Figures 9 (C) and (D) show the relationship between the log concentration of single-stranded DNA and the ΔRJP number for target-assisted synthesis of enzyme reporters (TASER) and invasive amplification-TASER (IA-TASER), respectively.

[0037] Figure 10 shows the sequence (5' → 3') of the gene construct ((A) of Figure 9) used in the study.

[0038] Figure 11 shows the results of analyzing the expression level of the gene construct ((A) of Figure 9) according to the cell-free expression time. The C-terminal His6-tag of the expressed protein was detected with an anti-His6-horseradish peroxidase conjugate.

[0039] Figure 12 is a schematic diagram illustrating a method for preparing sensor DNA for use in target-assisted synthesis of enzyme reporters (TASER) and the TASER method. (A) of Figure 12 shows a gene construct for expressing a reporter protein gene in a typical CFPS reaction. (B) shows sensor DNA having a single-stranded T7 promoter region. (C) shows a method for preparing the sensor DNA. The gene construct was synthesized by combining primers containing dUTP in the TBS and T7P regions, and the amplified polymerase chain reaction product was processed through a uracil-specific excision reaction (USER). (D) is a schematic diagram of a TASER experiment for detecting single-stranded nucleic acids.

[0040] Figure 13 schematically illustrates the reaction steps of an invasive amplification-TASER (IA-TASER). Figure 13 (A) illustrates the concept of an invasive amplification reaction using a pair of flap probes and invasive probes for detecting parvovirus as an example. The italicized A in the invasive probe represents an adenosine nucleotide added to improve cleavage efficiency. (B) indicates chemical modifications introduced into the flap probe. * represents a phosphorothioate bond, and bold letters represent 2'-O-methoxy-ethyl (2'MOE) modified nucleotides. (C) schematically illustrates a method for generating a reporter protein from a target nucleic acid using the IA-TASER.

[0041] Figure 14 shows the analysis of 16S rRNA transcripts of foodborne pathogens using IA-TASER and retroreflectance signal detection-based methods. (A); (B); (C) and (D) are the detection results for Escherichia coli, Salmonella enterica, Staphylococcus aureus, and Vibrio cholera, respectively.

[0042] Figure 15 shows the results confirming the detection specificity for S. Enterica of the detection method of the present invention, which was designed and set as the target nucleic acid 16S rRNA of S. Enterica. The 'Mixture' sample contains the 16S rRNA of four pathogens at the same concentration as the other test groups.

[0043] Figure 16 shows the analysis of the detection method of the present invention, which is designed and set as the target nucleic acid 16S rRNA of S. Enterica, applied to the total RNA purified from S. Enterica.

[0044] Figure 17 is a schematic diagram illustrating a process for detecting a target nucleic acid using the sensor DNA of the present invention. When the sensor DNA is designed by setting the indicated trigger ssDNA in Figure 17 as the target nucleic acid, the target nucleic acid can be detected without an invasive amplification (IA) reaction step.

[0045]

[0046] Detailed description of the invention and preferred embodiments

[0047] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Generally, the nomenclature used herein is well known and commonly used in the art.

[0048]

[0049] In the present invention, a detection technology for target nucleic acids based on cell-free protein synthesis and retroreflection signals was developed to overcome the limitations of existing technologies by quickly detecting target nucleic acids with high sensitivity using only simple optical detection devices such as cameras without advanced equipment.

[0050] In the present invention, a reporter protein including a first domain and a second domain that specifically bind to a first peptide attached to the surface of a retroreflective particle and a second peptide fixed to a substrate, respectively, is designed, and it is confirmed that the retroreflective particle can be fixed to the surface of the substrate using the reporter protein and a retroreflective signal can be generated, thereby detecting a target nucleic acid.

[0051] A sensor DNA comprising a sequence complementary to a target nucleic acid or a trigger nucleic acid, a promoter, and a sequence encoding the reporter protein was designed, and it was confirmed that the reporter protein was synthesized under a cell-free synthesis system only when the target nucleic acid was present in the sample (Fig. 17).

[0052] Therefore, the present invention, from one point of view,

[0053] i) a sequence complementary to a target nucleic acid or a trigger nucleic acid;

[0054] ii) promoter; and

[0055] iii) Sensor DNA for detecting a target nucleic acid comprising a sequence encoding a reporter protein,

[0056] The sequence complementary to the target nucleic acid or trigger nucleic acid and the promoter are single-stranded, and the sequence encoding the reporter protein is double-stranded or single-stranded,

[0057] The reporter protein comprises a first domain and a second domain,

[0058] The first domain specifically binds to the first peptide attached to the retroreflective particle,

[0059] The second domain relates to a sensor DNA for detecting a target nucleic acid that specifically binds to a second peptide fixed to a substrate.

[0060] A person skilled in the art will readily recognize that since the sequence complementary to the target nucleic acid or trigger nucleic acid of the present invention and the promoter are single-stranded, the sequence complementary to the target nucleic acid or trigger nucleic acid can hybridize with the target nucleic acid or trigger nucleic acid, and transcription cannot proceed if the promoter is single-stranded.

[0061] In one embodiment of the present invention, a sensor DNA comprising a sequence complementary to a target nucleic acid can be hybridized with the target nucleic acid, undergo a repair step using a DNA polymerase, and then be changed into a state capable of synthesizing a reporter protein according to a cell-free protein synthesis method.

[0062] In another embodiment of the present invention, a sensor DNA comprising a sequence complementary to a trigger nucleic acid can be hybridized with a trigger nucleic acid obtained from a target nucleic acid in a sample, and after a repair step by a DNA polymerase, can be changed into a state capable of synthesizing a reporter protein according to a cell-free protein synthesis method.

[0063] The term “target nucleic acid” in the present invention refers to a nucleic acid of interest to be detected, and a person skilled in the art will be able to design a target nucleic acid for detection of a sequence complementary to the target nucleic acid contained in the sensor DNA based on the present specification, according to the target nucleic acid.

[0064] The term "trigger nucleic acid" in the present invention refers to a nucleic acid selected as a detection target of sensor DNA to indirectly detect a target nucleic acid. By first performing a reaction to generate a trigger nucleic acid in the presence of a target nucleic acid, and then detecting the trigger nucleic acid using the sensor DNA of the present invention, the target nucleic acid can be indirectly detected.

[0065] An example of a reaction that generates a trigger nucleic acid in the presence of the target nucleic acid is an invasive amplification reaction (Victor I. Lyamichev, et al., Experimental and Theoretical Analysis of the Invasive Signal Amplification Reaction. Biochemistry 2000, 39 (31), 9523-9532.), which is incorporated herein by reference. However, in addition to the invasive amplification reaction, any technique that is available to those skilled in the art may be applied to the present invention without limitation.

[0066] The above invasive amplification reaction can be performed using a flap probe, an invasion probe, and an endonuclease. The flap probe comprises a sequence complementary to a target nucleic acid and a trigger nucleic acid sequence, and the invasion probe comprises a sequence complementary to a target nucleic acid sequence adjacent to the target nucleic acid sequence complementary to the flap probe (see FIG. 13). When a sample containing the target nucleic acid is hybridized with the flap probe and the invasion probe, a nucleic acid structure is formed in which a single-stranded trigger nucleic acid is ligated to the middle of a double-stranded nucleic acid, and the endonuclease recognizes the structure of the nucleic acid structure and cleaves the ligation to produce a trigger nucleic acid.

[0067] In particular, when using an invasive amplification reaction to obtain a trigger nucleic acid from a target nucleic acid, it is possible to design a pair of flap probes and invasive probes to generate trigger nucleic acids having the same sequence for target nucleic acids of various sequences, thereby changing the target nucleic acid without changing the sequence of the sensor DNA.

[0068] In the present invention, the sequence complementary to the target nucleic acid or trigger nucleic acid may be used without limitation as long as it can exist therein without interfering with the cell-free synthesis of the sensor DNA, and preferably includes 6 to 24 nucleic acids, more preferably 10 to 20 nucleic acids, and most preferably 14 to 16 nucleic acids, but is not limited thereto.

[0069]

[0070] In the present invention, the term "reporter protein" refers to a protein that is expressed when the sensor DNA of the present invention hybridizes with a target nucleic acid or a trigger nucleic acid, and generates a detectable signal to confirm the presence of the target nucleic acid. In particular, in the present invention, when the sensor DNA hybridizes with a target nucleic acid contained in a sample, a reporter protein can be synthesized, and the synthesized reporter protein generates a retroreflective signal through interaction with a retroreflective particle and a substrate.

[0071] In certain embodiments, the reporter protein comprises a first domain and a second domain, and the first domain and the second domain are any proteins that specifically bind to the first peptide and the second peptide attached / immobilized to the retroreflective particle and the substrate, respectively, without limitation.

[0072] From the description herein, it will be apparent to those skilled in the art that a reporter protein comprising a first domain and a second domain can immobilize a retroreflective particle on the surface of a substrate, thereby generating a retroreflective signal.

[0073] In this specification, terms such as first, second, etc. may be used to describe various components, but the components should not be limited by these terms. These terms are used only to distinguish one component from another.

[0074] The reporter protein may comprise a first domain - linker - second domain in order from the N'-terminus, or a second domain - linker - first domain in order from the N'-terminus.

[0075] In the present invention, the first domain and the second domain of the reporter protein may each include one or more identical proteins, and the number of proteins included in each domain may be changed to control specific binding affinity with the first peptide and the second peptide.

[0076] In the present invention, the reporter protein may be characterized as being a fusion protein.

[0077] The term “fusion protein” of the present invention generally refers to a recombinant protein that can be obtained by linking the genes of two or more identical or different proteins through cloning and then expressing them.

[0078] In the present invention, the sequence encoding the reporter protein may be a sequence of identical or different proteins, each constituting the first and second domains, linked through cloning. Those skilled in the art will be able to select an appropriate protein based on the description herein and clone its gene to recombinantly produce the reporter protein of the present invention.

[0079] For example, in the present invention, the first domain and the second domain of the reporter protein may be characterized as being MDM2 and Spycatcher003 (SC3), MDM2-MDM2 and Spycatcher003 (SC3), or Spycatcher003 (SC3) and MDM2, respectively, but are not limited thereto, and any protein capable of specifically binding to a specific peptide may be included in the first domain or the second domain of the present invention without limitation.

[0080] The above “MDM2” is a negative regulator of the tumor suppressor p53, and is a protein known to be overexpressed in cancer cells. MDM2 is particularly inhibited by PMI (N8A), which consists of 12 amino acids, and the dissociation constant (Kd) between MDM2 and PMI (N8A) is reported to be in the hundreds of picomolar range, demonstrating high binding affinity (Li, C., et al., J Mol Biol 2010, 398 (2), 200-13.).

[0081] In the present invention, “Spycatcher” is a protein constituting the SpyCatcher / SpyTag system based on a spontaneous isopeptide bond formation reaction between two fragments derived from the Streptococcus pyogenes fibronectin-binding protein FbaB40, together with the peptide “SpyTag.” In addition, “Spycatcher003” is a protein that specifically binds to “SpyTag003,” a peptide consisting of 16 amino acids, and is reported to exhibit a reaction approximately 400 times faster than the original isopeptide bond formation reaction (Zakeri, B., et al., Proc Natl Acad Sci USA 2012, 109 (12), E690-7.).

[0082]

[0083] For example, in the present invention, the sequence encoding the reporter protein may be characterized by including a nucleic acid sequence selected from SEQ ID NO: 1 to SEQ ID NO: 3.

[0084] In the present invention, the term "nucleic acid" is used interchangeably with "nucleotide," "nucleotide sequence," and "oligonucleotide." It may include a polymeric form of nucleotides of any length, deoxyribonucleotides or ribonucleotides, or analogs thereof. Polynucleotides may have any three-dimensional structure and may perform any known or unknown function. The polynucleotide is meant to comprehensively include DNA (gDNA and cDNA) and RNA molecules, and the basic structural unit, nucleotides, includes not only natural nucleotides but also analogs in which the sugar or base moiety is modified. The sequence of the polynucleotide may be modified. Such modifications include additions, deletions, or non-conservative or conservative substitutions of nucleotides.

[0085]

[0086] In the present invention, in order to synthesize a reporter protein from sensor DNA, the sequence encoding the reporter protein, the sequence complementary to the target nucleic acid or trigger nucleic acid, the promoter, the ribosome binding site that can be optionally added, and the terminator, etc. need to be operatively linked to other sequences.

[0087] "Operably linked" means a functional association between an expression regulatory sequence (e.g., a promoter, ribosome binding site, or terminator) and another nucleic acid sequence, and may be a gene and regulatory sequence(s) linked in such a way that gene expression is enabled when an appropriate molecule (e.g., a ribosome, a transcriptional activating protein) binds to the regulatory sequence(s), thereby causing the regulatory sequence to regulate transcription and / or translation of the other nucleic acid sequence.

[0088] The term "promoter" in the present invention refers to the upstream region of a gene involved in the initiation of transcription (the step of synthesizing RNA from DNA). That is, it refers to a site on a DNA strand where RNA polymerase binds or a site where RNA complementary to a DNA strand is synthesized, i.e., transcription begins.

[0089] As an example of an operatively connected, in the present invention, the sensor DNA may be characterized by including, in order from the 5' end to the 3' end, a sequence complementary to the target nucleic acid, a promoter, and a sequence encoding a reporter protein.

[0090] Therefore, in the present invention, the sensor DNA may additionally include a single-stranded or double-stranded ribosome binding site between the promoter and the sequence encoding the reporter protein to increase the certainty and efficiency of translation.

[0091] In the present invention, the sensor DNA may additionally include a terminator at the 3' end to increase the certainty and efficiency of transcription.

[0092] The term "terminator" of the present invention refers to a region of a gene involved in transcription termination. The terminator may include, but is not limited to, for example, the CaMV 35S terminator, nopaline synthase (NOS), rice α-amylase RAmy1 A terminator, phaseoline terminator, terminator of the Octopine gene of Agrobacterium tumefaciens, rrnB1 / B2 terminator of Escherichia coli, T7 terminator, etc.

[0093] In the present invention, the promoter may be characterized in that it is selected from the group consisting of a T3 promoter, a T5 promoter, a T7 promoter, a tac promoter, a trc promoter, a trp promoter, an arabinose promoter, a Lacuv5 promoter, and a LacI promoter.

[0094]

[0095] From another perspective, the present invention relates to a kit for detecting a target nucleic acid, comprising the sensor DNA.

[0096] In the present invention, the kit may further comprise a reaction solution for a cell-free protein synthesis system, and as an example, the kit may be characterized by further comprising DNA polymerase, RNA polymerase, ribosome, NTP mixture (ATP, TTP, GTP, and CTP), dNTP mixture (dATP, dTTP, dGTP, and dCTP), and amino acids. A person skilled in the art will be able to appropriately select and implement the composition of the reaction solution for a cell-free protein synthesis method and the concentrations of each component thereof.

[0097] In the present invention, “cell-free synthesis system” collectively refers to a method including a “cell-free synthesis method (cell-free or in vitro synthesis method),” a “cell-free peptide synthesis method (cell-free or in vitro peptide synthesis method),” a “cell-free protein synthesis method (cell-free or in vitro protein synthesis method),” or a “cell-free protein / peptide synthesis method” depending on the production target.

[0098] The term "cell-free protein synthesis system" of the present invention refers to a system that synthesizes proteins in vitro by adding substrates or enzymes to cell lysates or extracts and utilizing key elements (ATP (adenosine triphosphate), amino acids, etc.) required for protein synthesis. This cell-free protein synthesis method can overcome the shortcomings of existing protein production methods using cells, and cell-free protein synthesis extracts only the intracellular machinery and its factors related to protein production from cells, and artificially repeats only the protein synthesis process outside the cell while excluding the physiological regulatory mechanisms of the cell, thereby mass-producing the target protein in a short period of time, thereby enabling high-speed protein synthesis without going through a cell culture process. In addition, compared to the cell culture process in which protein expression proceeds within the space of the cell membrane and cell wall, it is a completely open method with no physical barriers, and has the advantage of being able to freely modify the conditions of protein synthesis for application to various studies. Additionally, it is expected that productivity can be increased because it can be free from various external conditions such as pH, temperature, and ionic strength when proteins are synthesized.

[0099]

[0100] In the present invention, the kit may further include a retroreflective particle to which a first peptide is attached and a substrate to which a second peptide is fixed.

[0101] In the present invention, the term “retroreflective particle” refers to a particle that exhibits the characteristic of retroreflecting light. The term “retroreflection” refers to a reflection in which light emitted from a light source is reflected on the surface of an object and returns toward the direction of the light source, and can be used interchangeably with the term “retroreflection.” When light is irradiated on a retroreflective particle, it has the characteristic of returning the light in the direction of the original light source regardless of the angle. In the present invention, the property of such retroreflective particles is utilized to irradiate light and the light returning to the light source is utilized as a signal to detect a target nucleic acid. Any particle that has the characteristic of retroreflecting light can be used in the present invention without limitation.

[0102] Known structures of retroreflective particles include cat's eye and corner reflector. The cat's eye has a structure in which a portion of a light-transmitting spherical particle's surface, preferably about half of the surface, is coated with a light-reflecting material, while the corner reflector typically has three orthogonal surfaces that reflect light.

[0103] In one embodiment of the present invention, retroreflective particles having the cat's eye structure were manufactured and used.

[0104] Therefore, in the present invention, the retroreflective particle may be characterized as being a spherical particle in which a portion of the surface of the core particle is coated with metal.

[0105] In light of the structure of the above retroreflective particles, it can be readily understood by those skilled in the art that the retroreflective particles retroreflect light incident from a side that is not coated with metal.

[0106] In order to achieve the object of the present invention, the retroreflective particle may be characterized in that 10% to 90%, preferably 30% to 70%, more preferably 40% to 60%, and most preferably 45% to 55% of the surface area of ​​the core particle is coated with metal.

[0107] Additionally, in the present invention, the core particle may be characterized by transmitting light.

[0108] In order to achieve the purpose of the present invention, it is preferable that the core particle is composed of a material that transmits light, and as an example, in the present invention, the core particle may be characterized by including a material selected from the group consisting of silica, glass, polystyrene, and poly(methyl methacrylate).

[0109] In order to achieve the purpose of the present invention, in the present invention, a portion of the surface of the core particle may be coated with a light-reflecting material, preferably a metal.

[0110] Additionally, in order to coat small-sized particles with a thin thickness, as an example, in the present invention, the metal may be characterized by including at least one selected from the group consisting of gold (Au), platinum (Pt), silver (Ag), and aluminum (Al).

[0111] In the present invention, the retroreflective particles may be characterized by having a diameter of 300 nm to 2000 nm, preferably 400 nm to 1500 nm, more preferably 500 nm to 1200 nm, and most preferably 600 nm to 1000 nm.

[0112] In the present invention, the first peptide may be characterized in that it is attached to a side of the core particle that is not coated with metal, and in this case, after the reporter protein binds to the first peptide and the second peptide attached / fixed to the retroreflective particle and the substrate, the retroreflective particle may be arranged so that the side that is not coated with metal faces the direction in which the substrate is located.

[0113] In the present invention, the first peptide may be characterized in that it is attached to the metal-coated side of the core particle, and in this case, after the reporter protein binds to the first peptide and the second peptide attached / fixed to the retroreflective particle and the substrate, the retroreflective particle may be arranged so that the side that is not coated with metal faces the opposite direction to the substrate.

[0114] When the first peptide is bound to the surface of the retroreflective particle that is not coated with metal, light is irradiated through the substrate based on the retroreflective particle to detect the retroreflective signal. Therefore, in the present invention, the substrate may be characterized as transmitting light.

[0115] As an example for achieving the purpose of transmitting light, in the present invention, the substrate may be characterized by including a material selected from the group consisting of silica, glass, polystyrene, and poly(methyl methacrylate).

[0116]

[0117] In the same way that the first domain and the second domain of the reporter protein can be used without limitation as long as they are proteins that specifically bind to the first peptide and the second peptide attached / immobilized to the retroreflective particle and the substrate, respectively, the first peptide and the second peptide of the present invention can be used without limitation as long as they are substances that specifically bind to the first domain and the second domain, respectively.

[0118] As an example, in the present invention, the first peptide and the second peptide may be characterized as being PMI (N8A) and SpyTag003 or SpyTag003 and PMI (N8A), respectively.

[0119]

[0120] In another aspect, the present invention relates to a method for detecting a target nucleic acid, comprising the following steps:

[0121] (a) a step of mixing a sample containing a target nucleic acid with the sensor DNA, hybridizing the target nucleic acid and the sensor DNA, and then treating the sample with a DNA polymerase to form a double-stranded nucleic acid;

[0122] (b) a step of synthesizing a reporter protein from the formed double-stranded nucleic acid;

[0123] (c) mixing the synthesized reporter protein with the retroreflective particles to which the first peptide is attached, and treating the substrate to which the second peptide is fixed, thereby arranging the retroreflective particles in a certain direction on the substrate; and

[0124] (d) A step of detecting a retroreflective signal generated from retroreflective particles by irradiating light.

[0125] In the present invention, the term “hybridization” refers to the formation of a double-stranded nucleic acid by hydrogen bonding between single-stranded nucleic acids having complementary base sequences, and is used in a similar sense to annealing. However, in a slightly broader sense, hybridization includes cases where the base sequences between two single-stranded nucleic acids are completely complementary (perfect match) as well as cases where some base sequences are not complementary (mismatch). In the present invention, it is obvious to those skilled in the art that the sequence complementary to the target nucleic acid of the sensor DNA is a site that binds to the target nucleic acid, and therefore, in order for hybridization to occur, the target nucleic acid and the sequence complementary to the target nucleic acid of the present invention must be completely complementary.

[0126] In the present invention, the DNA polymerase of step (a) may be characterized by polymerizing and extending a nucleic acid from the 3' end of the target nucleic acid hybridized with the sensor DNA.

[0127] DNA polymerases typically recognize the 3' end of damaged double-stranded nucleic acids and possess a DNA repair function that polymerizes and extends the nucleic acid from the 3' end. In the present invention, by utilizing this repair function, we designed a sensor DNA that allows DNA polymerase to form an intact double-stranded nucleic acid only in the presence of a target nucleic acid.

[0128] In the present invention, the "sample" includes various samples, and preferably, a biological sample is analyzed using the method of the present invention. More preferably, it may be a sample mixed with a virus species or a sample of an individual (e.g., a human, a mammal, a fish, etc.) infected with the virus, and biological samples of plant, animal, human, fungal, bacterial, and viral origin may be analyzed. When analyzing a sample of mammalian or human origin, the sample may be derived from a specific tissue or organ. Representative examples of tissues include connective, skin, muscle, or nervous tissue. Representative examples of organs include the eye, brain, lung, liver, spleen, bone marrow, thymus, heart, lymph, blood, bone, cartilage, pancreas, kidney, gallbladder, stomach, small intestine, testis, ovary, uterus, rectum, nervous system, gland, and internal blood vessels. The biological sample to be analyzed includes any cell, tissue, fluid, or other medium from a biological source that can be well analyzed by the present invention, including samples obtained from humans, animals, or food prepared for human or animal consumption. The biological sample to be analyzed also includes body fluid samples, including but not limited to blood, serum, plasma, lymph, breast milk, urine, feces, ocular fluid, saliva, semen, brain extracts (e.g., brain pulverizer), spinal fluid, appendix, spleen, and tonsil tissue extracts. Examples of such samples for which the present invention can be utilized include food samples collected from food prepared for human or animal consumption, wherein by detecting nucleic acids derived from foodborne pathogens in such food samples, it is possible to rapidly determine whether the food has been exposed to foodborne pathogens.

[0129] An additional step of extracting nucleic acids from the sample may be included, and the extraction of nucleic acids may be performed using, for example, various kits or extraction reagents that are commercially available and supplied.

[0130]

[0131] In the present invention, the step (b) may be characterized in that it is performed through a cell-free protein synthesis system.

[0132] Since the double-stranded nucleic acid formed according to step (a) of the present invention includes an intact promoter and a sequence encoding a reporter protein, the reporter protein can be synthesized from the double-stranded nucleic acid through a cell-free synthesis system.

[0133] In the present invention, the term "cell-free synthesis system" may be applied in the same manner as the description given above regarding the kit. The term "cell-free protein synthesis system" of the present invention refers to a method for synthesizing proteins in a test tube by adding a substrate or enzyme to cell lysates or extracts and utilizing key elements (ATP (adenosine triphosphate), amino acids, etc.) necessary for protein synthesis. This cell-free protein synthesis method can overcome the shortcomings of existing protein production methods using cells. Cell-free protein synthesis extracts only the intracellular machinery and its factors related to protein production from cells, and artificially repeats only the protein synthesis process outside the cell while excluding the physiological regulatory mechanisms of the cell, thereby mass-producing the target protein in a short period of time. Not only can it synthesize proteins at high speed without going through the cell culture process, but it is also a completely open method with no physical barriers compared to the cell culture process in which protein expression occurs within the space of the cell membrane and cell wall, and it has the advantage of being able to freely modify the protein synthesis conditions for application to various studies. Furthermore, because it is free from various external conditions such as pH, temperature, and ionic strength when protein is synthesized, it is expected to increase productivity.

[0134]

[0135] The reporter protein synthesized through the above step (b) includes a first domain, a second peptide, and a second domain. Therefore, the first domain of the reporter protein specifically binds to the first peptide attached to the retroreflective particle, and the second domain specifically binds to the second peptide fixed to the substrate, ultimately fixing the retroreflective particle to the substrate, and in the process, the retroreflective particle can be arranged in a certain direction on the substrate.

[0136] In the present invention, the retroreflective particle may be characterized as being a spherical particle in which a portion of the surface of the core particle is coated with metal.

[0137] In the present invention, when the first peptide is attached to the side of the core particle that is not coated with metal, after the reporter protein synthesized in step (b) binds to the first peptide and the second peptide attached / fixed to the retroreflective particle and the substrate, the retroreflective particle can be arranged so that the side that is not coated with metal faces the direction in which the substrate is located.

[0138] In the present invention, when the first peptide is bound to the metal-coated side of the retroreflective particle, after the reporter protein synthesized in step (b) binds to the first peptide and the second peptide attached / fixed to the retroreflective particle and the substrate, the retroreflective particle can be arranged so that the side not coated with metal faces the opposite direction to the substrate.

[0139] In the present invention, the term "retroreflective signal" refers to light that returns toward the light source due to the retroreflective phenomenon. In step (c), when light is irradiated on retroreflective particles arranged in a certain direction on the substrate, the light returns through the retroreflective phenomenon, and by detecting this, the target nucleic acid can be detected.

[0140]

[0141] In the present invention, the step (d) may be characterized by irradiating light in a direction that is not coated with metal of the retroreflective particles arranged in the step (c).

[0142] In the present invention, through the step (c), the retroreflective particles are arranged in a certain direction, and only when light is irradiated in a direction that is not coated with metal of the arranged retroreflective particles, the irradiated light is retroreflected and a retroreflective signal can be generated.

[0143]

[0144] In another aspect, the present invention relates to a method for detecting a target nucleic acid, comprising the following steps:

[0145] (a) a step of hybridizing a trigger nucleic acid obtained from a sample containing a target nucleic acid with a sensor DNA of claim 1 containing a sequence complementary to the trigger nucleic acid, and then treating the hybridized sensor DNA with a DNA polymerase to form a double-stranded nucleic acid;

[0146] (b) a step of synthesizing a reporter protein from the formed double-stranded nucleic acid;

[0147] (c) mixing the synthesized reporter protein with the retroreflective particles to which the first peptide is attached, and treating the substrate to which the second peptide is fixed, thereby arranging the retroreflective particles in a certain direction on the substrate; and

[0148] (d) A step of detecting a retroreflective signal generated from retroreflective particles by irradiating light.

[0149]

[0150] The detection method using sensor DNA containing a sequence complementary to the above trigger nucleic acid differs from the detection method containing a sequence complementary to the above target nucleic acid only in step (a) of forming a double-stranded nucleic acid, and the above-described contents can be equally applied to the remaining steps (b) to (d).

[0151] In the present invention, it is obvious to those skilled in the art that the sequence complementary to the trigger nucleic acid of the sensor DNA is a site that binds to the trigger nucleic acid, and therefore, in order for hybridization to occur, the trigger nucleic acid and the sequence complementary to the trigger nucleic acid of the present invention must be completely complementary.

[0152] When using a sensor DNA containing a sequence complementary to a trigger nucleic acid, the target nucleic acid can be indirectly detected by generating a trigger nucleic acid from a target nucleic acid and detecting the generated trigger nucleic acid using the sensor DNA.

[0153]

[0154] An example of a reaction that generates a trigger nucleic acid in the presence of the target nucleic acid is an invasive amplification reaction (Victor I. Lyamichev, et al., Experimental and Theoretical Analysis of the Invasive Signal Amplification Reaction. Biochemistry 2000, 39 (31), 9523-9532.), which is incorporated herein by reference. However, in addition to the invasive amplification reaction, any technique that is available to those skilled in the art may be applied to the present invention without limitation.

[0155] The above invasive amplification reaction can be performed using a flap probe, an invasion probe, and an endonuclease. The flap probe includes a sequence complementary to a target nucleic acid and a trigger nucleic acid sequence, and the invasion probe includes a sequence complementary to a target nucleic acid sequence adjacent to the target nucleic acid sequence complementary to the flap probe (see FIG. 13). When a sample containing the target nucleic acid is hybridized with the flap probe and the invasion probe, a nucleic acid structure is formed in which a single-stranded trigger nucleic acid is ligated to the middle of a double-stranded nucleic acid, and the endonuclease recognizes the structure of the nucleic acid structure and cleaves the ligation to produce the trigger nucleic acid.

[0156] In particular, when using an invasive amplification reaction to obtain a trigger nucleic acid from a target nucleic acid, it is possible to design a pair of flap probes and invasive probes to generate trigger nucleic acids having the same sequence for target nucleic acids of various sequences, thereby changing the target nucleic acid without changing the sequence of the sensor DNA.

[0157] Table 7 of Example 5 below is an example of flap probes and invasion probes designed according to target nucleic acids. It is readily apparent to those skilled in the art that, based on the description herein, flap probes and invasion probes capable of generating trigger nucleic acids from various target nucleic acids, including but not limited to, are capable of being designed.

[0158] In the present invention, the DNA polymerase of step (a) may be characterized by polymerizing and extending a nucleic acid from the 3' end of the target nucleic acid hybridized with the sensor DNA.

[0159] DNA polymerases typically recognize the 3' end of damaged double-stranded nucleic acids and possess a DNA repair function that polymerizes and extends the nucleic acid from the 3' end. In the present invention, by utilizing this repair function, we designed a sensor DNA that allows DNA polymerase to form a complete double-stranded nucleic acid only in the presence of a trigger nucleic acid.

[0160]

[0161] In the present invention, the target nucleic acid may be characterized as being a nucleic acid derived from a food poisoning pathogen, but is not limited thereto, and a person skilled in the art can appropriately select the target nucleic acid depending on the purpose of detection based on the description herein.

[0162] In one embodiment of the present invention, a sensor DNA capable of detecting nucleic acids derived from food poisoning pathogens, particularly 16S rRNA, was designed, and it was confirmed that detection of 16S rRNA nucleic acids was possible according to the method of the present invention, and the 16S rRNA was specifically detected even in a sample containing total RNA extracted / purified from the food poisoning pathogen. By setting the nucleic acids derived from the food poisoning pathogens as a detection target, it is possible to determine whether the food poisoning pathogens are present in a sample.

[0163] In one embodiment of the present invention, a pair of sensor DNA, flap probe, and invasion probe capable of detecting 16S rRNA of Escherichia coli, Salmonella enterica, Staphylococcus aureus, and Vibrio cholera, which are known as food poisoning pathogens, were designed, and 16S rRNA derived from the food poisoning pathogens was successfully detected.

[0164]

[0165] Hereinafter, the present invention will be described in detail with examples and the like to aid understanding. However, the examples according to the present invention may be modified in various different forms, and the scope of the present invention should not be construed as being limited to the following examples.

[0166]

[0167] Example 1: Preparation and purification of reporter protein, first peptide, and second peptide

[0168] Example 1-1: Plasmid construction

[0169] The synthetic gene for MDM2-SpyCatcher003 (Table 1) was cloned into pET21a using the NdeI and XhoI sites, generating pSPEL870. To construct MDM2-MDM2-SpyCatcher003, an additional MDM2 gene was amplified using primers 1 and 2 (Table 2) and inserted into pSPEL870 between MDM2 and SpyCatcher003 using the NheI and HindIII sites, generating pSPEL1019. The synthetic gene for the SpyTag003-Z domain (Table 1) was cloned into pET21a using the BamHI and XhoI sites, generating pSPEL860. To add a BamHI site to the C-terminus of MDM2-SpyCatcher in pSPEL870, the gene was amplified using primers 3 and 4 (Table 2) and inserted into pSPEL870 using NdeI and XhoI sites to generate pSPEL901. The GFP(11.3.3) gene was amplified using primers 5 and 6 (Table 2) from pQE80L-GFP(11.3.3)31 and cloned into pSPEL901 using BamHI and XhoI sites to generate pSPEL902. To prepare target-assisted synthesis sensor DNA plasmids for target-assisted enzyme reporter synthesis (TASER) and invasive amplification-TASER (IA-TASER), MDM2-MDM2-SpyCatcher003 was cloned into pK732 using NdeI and SalI sites to generate pSPEL1301. The gene was synthesized using Integrated DNA Technologies (USA). All primers were synthesized by Macrogen (Seoul, Korea).

[0170] The gene base sequences and primer sequences used in the present invention are shown in Tables 1 and 2, respectively.

[0171]

[0172]

[0173]

[0174] Example 1-2: Protein expression and purification

[0175] Expression of MDM2-SpyCatcher003, MDM2-MDM2-SpyCatcher003, and MDM2-SpyCatcher003-GFP used in the examples was performed as follows:

[0176] E. coli BL21(DE3) cells transformed with pSPEL870, pSPEL1019, or pSPEL902 were used to express MDM2-SpyCatcher003, MDM2-MDM2-SpyCatcher003, or MDM2-SpyCatcher003-GFP, respectively. The recombinant strains were cultured in 500 mL 2xYT at 37°C until the optical density (OD600) reached 0.5, at which point protein expression was induced overnight at 20°C with 0.2 mM isopropyl β-D-1-thiogalactopyranoside (IPTG, Bioshop, Canada). Cells were harvested by centrifugation (9300 × g for 15 min) and stored at -20°C until purification. Proteins with a C-terminal His6 tag were purified using Ni-NTA resin (Qiagen, Germany) according to the manufacturer's protocol. The protein solution was buffer-exchanged into phosphate-buffered saline (PBS; 10 mM potassium phosphate, 150 mM NaCl, pH 7.4) using a centrifugal filter device (MWCO: 10000, Merck Millipore, USA).

[0177] The SpyTag003-Z domain peptide used in the examples was prepared by the following method:

[0178] The SpyTag003-Z domain was expressed using E. coli BL21(DE3) cells transformed with pSPEL860. The recombinant strain was cultured in 250 mL 2xYT at 37°C until an OD600 of 0.5 was reached. Protein expression was induced with 1 mM IPTG at 37°C for 6 h. Cells were harvested by centrifugation (9300 × g for 15 min) and stored at -20°C until purification. The N-terminal His6-tagged protein was purified using Ni-NTA resin according to the manufacturer's protocol. The SpyTag003-Z domain solution was exchanged with PBS using a centrifugal filter device (MWCO: 10000).

[0179] The concentration of purified protein was determined by measuring the absorbance at 280 nm using the extinction coefficient calculated from the ProtParam site (https: / web.expasy.org / protparam / ).

[0180]

[0181] Example 1-3: Cell-free protein synthesis (CFPS) method

[0182] For cell-free protein synthesis, 7.2 μL of a solution containing reporter protein was mixed with 36 μL of a mixture solution with the following composition:

[0183] HEPES-KOH (pH 7.5) 57 mM; ATP 1.2 mM; CTP, GTP, and UTP each 0.85 mM; DL-dithiothreitol 2 mM; E. coli total tRNA mixture (from strain MRE600) 0.17 mg / mL; cAMP 0.64 mM; potassium glutamate 90 mM; ammonium acetate 80 mM; magnesium acetate 12 mM; 1-5-formyl-5,6,7,8-tetrahydrofolic acid (folic acid) 34 μg / mL; 1.5 mM each of the 20 amino acids; creatine phosphate 67 mM; creatine kinase 3.2 μg / mL; E. coli DNA polymerase I 0.2 U / μL; dNTP 0.25 mM; and 26% (v / v) of S12 extract prepared from E. coli BL21(DE3)Star (Kim, T.-W., et al., Biotechnology and Bioprocess Engineering 2008, 13 (4), 464-469.). The mixture was incubated in a microtube shaker (1000 rpm) at 30°C for 1 hour.

[0184]

[0185] Example 1-4: Microscale thermophoresis

[0186] The dissociation constant (Kd) between MDM2 and PMI(N8A) peptide was determined using a NanoTemper® Monolith NT.115 (NanoTemper Technologies, Germany). MDM2-SpyCacher003 was labeled with the second-generation dye RED-tris-NTA (NanoTemper Technologies, Germany). The labeled proteins were incubated with various concentrations of PMI(N8A) (600 pM–20 μM), and the mixture was loaded onto standard-treated capillaries (NanoTemper Technologies, Germany). All experiments were repeated at least three times. Data were analyzed using MO.Affinity Analysis software version 3.0.4 (NanoTemper Technologies).

[0187]

[0188] Example 2: Preparation of reporter protein and confirmation of binding with first and second peptides

[0189] Example 2-1: Design of a reporter protein having a first domain and a second domain

[0190] In a conventional cell-free synthesis system for synthesizing and measuring reporter proteins, a reporter protein comprising a first domain and a second domain was designed to address the issue of incompatibility of retroreflective signals. Protein-peptide interactions were utilized to design a reporter protein capable of binding to both retroreflective particles and substrates.

[0191] When utilizing protein-peptide interactions, peptides have the advantage of being relatively simple to introduce orthogonal reactive groups at specific positions, enabling directional fixation of the peptide to the surface through the reactive groups, and providing higher surface stability upon modification compared to when using proteins.

[0192] A reporter protein and a first peptide and a second peptide were designed using MDM2-PMI (N8A) and SpyCatcher003-SpyTag00337, which are previously reported protein-peptide interactions (Li, C., et al., J Mol Biol 2010, 398 (2), 200-13.).

[0193] The reporter protein used in this example is a fusion protein of MDM2 and SpyCatcher003. Because the N- and C-termini of both proteins are located in close proximity, the fusion sequence is expected not to interfere with binding to the PMI (N8A) and SpyTag003 peptides (Figure 2).

[0194] Reporter proteins (MDM2-SpyCatcher003, SpyCatcher003-MDM2, MDM2-MDM2-SpyCatcher003) were prepared according to the method of Example 1, and their expression in a cell-free synthetic system was tested. The sequences of the prepared reporter proteins are shown in Table 3. In the reporter proteins, the linkers connecting MDM2 and MDM2, and MDM2 and SpyCater003 are underlined:

[0195]

[0196]

[0197] As a result, it was confirmed that all of the above reporter proteins were expressed under the cell-free synthesis system, and in particular, the expression level of MDM2-SpyCatcher003 was even higher. The MDM2-MDM2-SpyCatcher003 fusion protein, which is MDM2-SpyCatcher003 that additionally contains MDM2 at the N-terminus, also showed high expression (Fig. 3A).

[0198]

[0199] Example 2-2: Confirmation of binding between reporter protein and first and second peptides

[0200] Among the above fusion proteins, MDM2-SpyCatcher003, which had a higher expression level, was purified, mixed with the SpyTag003-Z domain, and electrophoresed to confirm that the MDM2-SpyCatcher003 and SpyTag003-Z domains bind to each other (Fig. 3B).

[0201] After expressing and purifying MDM2-SpyCatcher003 according to the method of Example 1-2, the Kd value between MDM2-SpyCatcher003 and PMI (N8A) was measured according to the microscale thermophoresis (MST) method of Example 1-4. As a result, the Kd value between the protein and PMI (N8A) was confirmed to be approximately 500 nM (Fig. 3C). When MDM2-SpyCatcher003 was cell-free synthesized according to the method of Example 1-3, the Kd value was also confirmed to be approximately 500 nM (Fig. 4). In addition, MDM2-MDM2-SpyCatcher003, which is expected to have a higher binding affinity to PMI (N8A), was constructed. As a result, MDM2-MDM2-SpyCatcher003 produced in CFPS showed higher expression (Fig. 3A). The above results confirm that MDM2 and SpyCatcher003 contained in the reporter protein fused with MDM2 and SpyCatcher003 still maintain specific binding affinity with PMI (N8A) and Spy tag003, respectively.

[0202]

[0203] Example 3: Detection of reporter proteins using retroreflective particles and substrates with attached peptides.

[0204] The silica surface of the retroreflective particle and the glass surface of the substrate were each attached / immobilized with the first peptide and the second peptide using the following four-step method (Fig. 5):

[0205] 1) APTES treatment step to introduce primary amine groups

[0206] 2) Step of reacting amine and NHS-PEG5-Tris-PEG3-Azide

[0207] 3) Transformation-promoting click reaction step between the surface azide and DBCO-PEG4-DBCO; and

[0208] 4) Immobilization step of peptide having azido group.

[0209] As the first peptide and the second peptide, PMI (N8A) and SpyTag003, which specifically bind to MDM2 and SpyCatcher003 of the reporter proteins used in Example 2, respectively, were used.

[0210] The amino acid sequences of the PMI (N8A) and SpyTag003 peptides used in this example are as shown in Table 4 below:

[0211]

[0212]

[0213] Example 3-1: Attachment of the first peptide to the surface of a retroreflective particle

[0214] Retroreflective particles were fabricated by sequential metal evaporation of aluminum and gold layers onto 700 nm SiO2 microparticles (Han, YD, et al., ACS Appl Mater Interfaces 2016, 8 (17), 10767-74.). Spherical particles partially surface-coated with metal exhibited the property of retroreflecting light.

[0215] Attachment of the first peptide (SpyTag003) to the above retroreflective particle was performed through the following steps:

[0216] Primary amine groups were introduced to the silica surface by treating 1 mg / ml RJP with 0.1% 3-aminopropyltriethoxysilane (APTES; Sigma-Aldrich, USA) in the dark for 1 h. The particles were washed with dimethyl sulfoxide (DMSO; Sigma-Aldrich, USA) by centrifugation and resuspension, and the amine groups of RJP were reacted with 1 mM NHS-PEG5-Tris-PEG3-Azide (Conju-probe, USA) in DMSO for 30 min. Unreacted NHS esters were quenched with 10 mM ethanolamine (EA; Sigma-Aldrich) for 30 min, and the particles were washed with DMSO. The azide groups of RJP were reacted with 5 mM DBCO-PEG4-DBCO (Broadfarm, USA) for 3 h. After washing with DMSO and PBS, RJPs were incubated overnight at 25°C with 10 μM SpyTag003-azide peptide (Anygen, Korea) dissolved in PBS. After washing with PBS, the particles were blocked with 1% bovine serum albumin (BSA) in PBS for 2 h. Peptide-modified RJPs were stored in PBS at 4°C.

[0217]

[0218] Example 3-2: Immobilization of a second peptide on a substrate surface

[0219] A glass chip (Paul Marienfeld, Germany) was used as the substrate. The substrate surface was treated with 0.1% APTES for 1 h in a darkroom to introduce primary amine groups onto the silica surface. After washing the glass surface with DMSO, the amine groups on the surface were reacted with 1 mM NHS-PEG-Tris-PEG-3-Azide in DMSO for 30 min. Unreacted NHS esters were quenched with 10 mM EA for 30 min, and the surface was washed with DMSO. The azide groups on the glass surface were reacted with 5 mM DBCO-PEG4-DBCO for 3 h. After washing with DMSO and PBS, polydimethylsiloxane (PDMS) channels prepared according to a previously described method (12) were fixed to the chip glass surface. The inner surface was incubated overnight at 25°C with 10 μM azide-PMI (N8A) peptide (Anygen, Korea) dissolved in PBS. After washing with PBS, the surface was blocked for 2 hours by adding 1% BSA in PBS.

[0220]

[0221] Example 3-3: Confirmation of attachment / anchoring of first and second peptides to retroreflective particles and substrates and detection of reporter proteins

[0222] PMI(N8A) and SpyTag003 were synthesized with azide-lysine at the N- and C-termini, respectively. The positions of the unnatural amino acids were determined based on the structures of MDM2-PMI(N8A) (PDB: 3LNZ36) and SpyCatcher-SpyTag (PDB: 4MLI43) (Fig. 6). Modification of retroreflective particles and substrate surfaces using peptides was evaluated using MDM2-SpyCatcher003-GFP (Fig. 7). Fluorescence images of retroreflective particles with SpyTag003 attached showed that half of the particles were stained with the fluorescent protein (Fig. 7A), and fluorescence signals were observed on the substrate only when PMI(N8A) was immobilized on the substrate surface (Fig. 7B). The reporter protein (MDM2-MDM2-SpyCatcher003) was expressed and purified according to the method of Example 1-2, and the retroreflective particle to which the first peptide (SpyTag003) was attached and the substrate to which the second peptide (PMI (N8A)) was fixed were treated as follows:

[0223] After incubating retroreflective particles and reporter proteins attached to the surface of SpyTag003 at various concentrations, the mixture was applied to a chip immobilized with PMI (N8A). A concentration-dependent relationship was observed between the reporter protein and retroreflective particles (LOD 8.17 nM, Figure 8). This result demonstrates that the detection method utilizing the reporter protein, retroreflective particles, and substrate of the present invention works well and has excellent sensitivity.

[0224]

[0225] Example 4: Design of sensor DNA for target nucleic acid detection

[0226] Example 4-1: Design of a Gene Construct for Synthesizing a Reporter Protein

[0227] A reporter protein was synthesized using a gene construct (Figures 9A and 10) containing a target nucleic acid binding site (TBS)-T7 promoter (T7P)-ribosome binding site (RBS)-reporter protein sequence (RPS)-T7 terminator (T7T). The TBS sequence was designed to hybridize with the single-stranded nucleotide T7UP. The base sequence of the constructed gene construct is shown in Figure 10.

[0228] PCR fragments of the gene construct were cultured for various periods of time in a cell-free protein expression system to synthesize reporter proteins through integrated transcription and translation. After 60 minutes, the amount of protein stopped increasing (Fig. 11). The expressed reporter protein was analyzed using a retroreflective signal according to the method of Example 3. Cell-free protein synthesis was performed for various concentrations of the gene construct, and a concentration-dependent relationship was observed in the range of 100 fM-1 nM (Fig. 9B). The LOD was confirmed to be 92.7 fM, which is approximately 100,000-fold lower than the LOD based on the purified reporter protein. This result indicates that the cell-free synthesis step converts the target nucleic acid into a reporter protein and ultimately amplifies the retroreflective signal.

[0229]

[0230] Example 4-2: Structural design of sensor DNA that synthesizes reporter protein in the presence of target nucleic acid

[0231] Target-assisted synthesis of enzyme reporters (TASER) is a method for detecting target nucleic acids using a cell-free protein synthesis system (CFPS). To implement the TASER method, which synthesizes reporter proteins only in the presence of target nucleic acids, a sensor DNA was designed based on the gene construct of Example 4-1, with a single-stranded structure for the target nucleic acid binding site and T7 promoter (T7P) region (Figure 12B). The method is as follows:

[0232] The MDM2-MDM2-SpyCatcher003 gene from pSPEL1301 was amplified using primers 7 and 8 (Table 5). Primer 7, which contains sequences for the target-binding site (TBS) and T7 promoter (T7P), was synthesized using dUTP for the subsequent uracil-specific excision reaction (USER). The PCR product was incubated with EZ™ USE Enzyme (Enzynomics, Korea) for the USER reaction according to the manufacturer's protocol, and the sensor DNA was purified using the Wizard ® SV Gel and PCR Clean-Up system (Promega, USA).

[0233]

[0234]

[0235] PCR was performed using a primer containing forward uracil, and the product was subjected to a uracil-specific excision reaction (USER) to expose single-stranded TBS and T7P (Fig. 12C). Double-stranded T7P is required for transcription by T7 RNA polymerase, and reporter protein synthesis through CFPS cannot be performed when only sensor DNA is present. Only when the target nucleic acid (T7UP) binds to the TBS of the template, DNA polymerase I present in the CFPS system converts the template into a gene construct suitable for transcription, and T7 RNA polymerase transcribes the gene construct.

[0236] T7UP at various concentrations was incubated with the template, and the mixture was transferred to a CFPS solution. The synthesized reporter protein was analyzed using an RJP-based sensing method. A concentration-dependent relationship of 13.6 fM was observed with an LOD of T7UP ranging from 100 fM to 1 nM (Figure 9C). These results demonstrate the successful integration of the developed RJP-based sensing strategy with the TASER reaction, which translates the presence of target nucleic acids into reporter protein synthesis.

[0237] The sensor DNA construct consists of a target-binding site (TBS), a T7 promoter (T7P), a ribosome-binding site (RBS), a reporter protein sequence (RPS), and a T7 terminator (T7T; Figure S1A). The polymerase chain reaction product, synthesized and amplified by the ligation of a dUTP-containing primer to the TBS and T7P regions, was processed through a uracil-specific excision reaction (USER) ( Figure 12C ). The product was used as the sensor DNA for the TASER method. If the T7 promoter is single-stranded, transcription by T7 RNA polymerase does not occur. When the target nucleic acid hybridizes with TBS, DNA polymerase I in the CFPS mixture converts the sensor DNA to a double-stranded form. T7 DNA polymerase transcribes the sensor DNA, and the reporter protein is synthesized ( Figure 12D ).

[0238]

[0239] Example 4-3: Design of a detection method including an additional invasive signal amplification reaction step

[0240] Instead of changing the TBS for each target sequence, we implemented an additional invasive amplification (IA) step that converts the target nucleic acid into T7UP (Fig. 13). The IA reaction involves an endonuclease, such as FEN1 or Tth, that cleaves the junction formed between double-stranded and single-stranded nucleic acids (Fig. 13A). By introducing the IA reaction, the TASER method was implemented by designing invasive probes and flap probes according to the target nucleic acid without having to change the sensor DNA structure for each target nucleic acid (Fig. 13C). A pair of invasive probes and flap probes was designed for each target, and the IA reaction generated a trigger nucleic acid that binds to the TBS of the sensor DNA. Since multiple copies of a single-stranded nucleic acid are generated for a single target nucleic acid, an amplification effect through the IA step is also expected. To reduce background signal, two additional modifications (a phosphorothioate bond and a 2'O-methoxy-ethyl modification to the base) were introduced at the 3' end of the flap probe (Fig. 13B).

[0241] Experiments were conducted to verify the effectiveness of the additionally introduced IA reaction step. IA probe pairs were designed to form junctions between double-stranded and single-stranded nucleic acids in the presence of target nucleic acids (44-45). When the target nucleic acid and both the flap probe and invasion probe hybridize, endonucleases such as FEN1 and Tth recognize and cleave the flap probe, and the resulting trigger nucleic acid (T7UP) triggers the synthesis of the reporter protein. A chemically synthesized sequence of canine parvovirus (CPV2a) DNA (4341-4470) was used as the target sequence. The IA step was applied to various concentrations of CPV2a target nucleic acids. The products were used in the TASER reaction and analyzed using the retroreflective signal detection method described in Example 3. A concentration-dependent relationship was observed with an LOD of 28.2 fM for the CPV2a target nucleic acid (Fig. 9D). The above results indicate that the simple and sensitive nucleic acid detection method designed by utilizing the efficient signal transmission and retroreflection signal detection method of the TASER detection method introducing IA works well.

[0242]

[0243] Example 5: Confirmation of detection of 16S rRNA derived from food poisoning pathogen using the method of the present invention

[0244] The detection method of the target nucleic acid identified in Example 4 above was applied to the detection of 16S rRNA of pathogens containing both conserved and variable regions. 16S rRNA has been used to identify bacterial species and generally targets the fourth variable domain. First, the 16S rRNA sequences of the pathogens to be detected (Escherichia coli, Salmonella enterica, Staphylococcus aureus, and Vibrio cholera) were compared (Table 6), and pairs of flap probes and invasion probes for each target were designed (Table 7) according to previously reported general guidelines (Li, L., et al., J Mol Biol 2014, 426 (2), 309-17.; V Lyamichev, et al., Nat Biotechnol. 1999, 17 (3), 292-6.). The RNA sequences of the above four pathogens and the base sequences of their probe pairs are shown in Tables 6 and 7 below.

[0245]

[0246]

[0247]

[0248]

[0249] In Table 6 above, the sequence complementary to the flap probe is indicated in bold, and the sequence complementary to the invasion probe is indicated in underline.

[0250]

[0251] In Table 7 above, the underlined portion represents the T7UP sequence capable of hybridizing with the sensor DNA. * indicates a phosphorothioate bond. Bold letters indicate nucleotides with a 2'-O-methoxy-ethyl (2'MOE) modification. Additionally, the adenosine at the 3' end of the invasion probe is a nucleotide added to improve cleavage efficiency.

[0252] The target 16S rRNA molecule of the pathogen was synthesized through transcription and analyzed using the method of the present invention, which includes the generation of a trigger product through the invasive amplification reaction of Example 4, detection of the target nucleic acid, and a retroreflective signal of Example 3. The target 16S rRNA was synthesized using the following method:

[0253] Synthetic 16S rRNA genes for target pathogens (Vibrio cholerae, Salmonella enterica, Escherichia coli, and Staphylococcus aureus; Table 1) were cloned into the pTOP vector using the TOPcloner™ PCR Cloning kit (Enzynomics, Korea). The resulting plasmids were amplified using primers 9 and 10, 11, 12, or 13 (Table 8), and the amplified products were transcribed using T7 RNA polymerase (Thermo Fisher Scientific, USA). The reaction mixtures were purified using the Qiagen RNeasy Mini Kit (Qiagen, Germany). The genes for 16S rRNA were synthesized by Integrated DNA Technologies (USA).

[0254]

[0255] A concentration-dependent relationship was observed in the range of 5 fM to 50 pM for four target 16S rRNA, and the LOD was identified as a single-digit femtomolar concentration (E. coli, 1.24 fM; S. enterica, 1.10 fM; S. aureus, 0.99 fM; V. cholera, 0.43 fM; Fig. 14). The specificity of the 16S rRNA detection method for S. Enterica was evaluated by comparing it with the test results for RNA of other pathogens (Fig. 15). S. Enterica is one of the most common foodborne pathogens (Kim, S.; et al., J Microbiol Biotechnol 2017, 27 (11), 1983-1993.). As a result of the experiment, retroreflective signals were observed only in samples containing 16S rRNA of S. Enterica.

[0256] Additionally, the detection method of the present invention was applied to purified total RNA of S. Enterica (Fig. 16). As a result, 1.3 x 10 -1 CFU / μL to 1.3 x 10 4 A concentration-dependent relationship was observed in the CFU / μL range, with an LOD of 3.8 x 10 -1 CFU / μL. This result indicates that the detection method of the present invention, which sets the 16S rRNA of the target pathogen as the target nucleic acid, can detect food poisoning pathogens.

[0257]

[0258] The method for detecting a target nucleic acid using sensor DNA according to the present invention is based on a cell-free protein synthesis system and a retroreflective signal, and can detect a target nucleic acid with very high sensitivity, such as having a limit of detection (LOD) of several femtomoles (fM), and can detect a target nucleic acid using only a simple optical detector such as a camera, so that the target nucleic acid can be detected more easily than conventional target nucleic acid detection methods based on fluorescence, bioluminescence, and electrochemical signals.

[0259] In addition, the method for detecting a target nucleic acid according to the present invention has the advantage of being able to easily change the target nucleic acid by changing the sequence complementary to the target nucleic acid of the sensor DNA or changing the sequence of a probe pair for obtaining a trigger nucleic acid from the target nucleic acid.

[0260]

[0261] While specific aspects of the present invention have been described in detail above, it will be apparent to those skilled in the art that these specific statements are merely preferred embodiments and do not limit the scope of the present invention. Therefore, the substantial scope of the present invention is defined by the appended claims and their equivalents.

[0262]

[0263] Electronic file attached.

Claims

1. i) a sequence complementary to a target nucleic acid or a trigger nucleic acid; ii) Promoter; and iii) a sensor DNA for detecting a target nucleic acid comprising a sequence encoding a reporter protein, The sequence complementary to the target nucleic acid or trigger nucleic acid and the promoter are single-stranded, and the sequence encoding the reporter protein is double-stranded or single-stranded, The reporter protein comprises a first domain and a second domain, The first domain specifically binds to the first peptide attached to the retroreflective particle, The second domain is a sensor DNA for detecting a target nucleic acid that specifically binds to a second peptide fixed to a substrate.

2. A sensor DNA according to claim 1, characterized in that the reporter protein is a fusion protein.

3. In the first paragraph, the first domain and the second domain of the reporter protein are MDM2 and Spycatcher003(SC3), MDM2-MDM2 and Spycatcher003(SC3), or Spycatcher003(SC3) and MDM2, respectively.

4. In the third paragraph, a sensor DNA comprising a sequence encoding the reporter protein and a nucleic acid sequence selected from SEQ ID NO: 1 to SEQ ID NO:

3.

5. A sensor DNA comprising, in order from the 5' end to the 3' end, a sequence complementary to the target nucleic acid, a promoter, and a sequence encoding a reporter protein.

6. A sensor DNA according to claim 5, further comprising a single-stranded or double-stranded ribosome binding site between the promoter and the sequence encoding the reporter protein.

7. A sensor DNA according to claim 5, further comprising a terminator at the 3' end.

8. A sensor DNA for detecting a target nucleic acid, wherein the promoter in paragraph 1 is selected from the group consisting of a T3 promoter, a T5 promoter, a T7 promoter, a tac promoter, a trc promoter, a trp promoter, an arabinose promoter, a Lacuv5 promoter, and a LacI promoter.

9. A kit for detecting a target nucleic acid, comprising the sensor DNA of any one of claims 1 to 8.

10. A kit for detecting a target nucleic acid, further comprising a DNA polymerase, an RNA polymerase, a ribosome, an NTP mixture (ATP, TTP, GTP, and CTP), a dNTP mixture (dATP, dTTP, dGTP, and dCTP), and an amino acid in the 9th paragraph.

11. A kit for detecting a target nucleic acid, further comprising a retroreflective particle having a first peptide attached thereto and a substrate having a second peptide fixed thereto in accordance with claim 9.

12. A kit for detecting a target nucleic acid, characterized in that in claim 11, the retroreflective particle is a spherical particle in which a portion of the surface of the core particle is coated with metal.

13. A kit for detecting a target nucleic acid, characterized in that the core particle in claim 12 transmits light.

14. A kit for detecting a target nucleic acid, wherein the core particle comprises a material selected from the group consisting of silica, glass, polystyrene, and poly(methyl methacrylate).

15. A kit for detecting a target nucleic acid, wherein in paragraph 12, the metal comprises at least one selected from the group consisting of gold (Au), platinum (Pt), silver (Ag), and aluminum (Al).

16. A kit for detecting a target nucleic acid, wherein the retroreflective particles in paragraph 12 are particles having a diameter of 300 nm to 2000 nm.

17. A kit for detecting a target nucleic acid, characterized in that in claim 12, the first peptide is attached to a side of the core particle that is not coated with metal.

18. A kit for detecting a target nucleic acid, characterized in that the substrate in claim 11 transmits light.

19. A kit for detecting a target nucleic acid, wherein the substrate comprises a material selected from the group consisting of silica, glass, polystyrene, and poly(methyl methacrylate).

20. A kit for detecting a target nucleic acid, characterized in that in claim 11, the first peptide and the second peptide are PMI (N8A) and SpyTag003 or SpyTag003 and PMI (N8A), respectively.

21. A method for detecting a target nucleic acid comprising the following steps: (a) a step of mixing a sample containing a target nucleic acid with the sensor DNA of claim 1, hybridizing the target nucleic acid and the sensor DNA, and then treating with a DNA polymerase to form a double-stranded nucleic acid; (b) a step of synthesizing a reporter protein from the formed double-stranded nucleic acid; (c) mixing the synthesized reporter protein with the retroreflective particle to which the first peptide is attached, and treating the substrate to which the second peptide is fixed, thereby arranging the retroreflective particle in a certain direction on the substrate; and (d) A step of detecting a retroreflective signal generated from a retroreflective particle by irradiating light.

22. A method for detecting a target nucleic acid in claim 21, characterized in that the DNA polymerase of step (a) polymerizes and extends a nucleic acid from the 3' end of the target nucleic acid hybridized with the sensor DNA.

23. A method for detecting a target nucleic acid, characterized in that the step (b) in claim 21 is performed through a cell-free protein synthesis system.

24. A kit for detecting a target nucleic acid, characterized in that in claim 21, the retroreflective particle is a spherical particle in which a portion of the surface of the core particle is coated with metal.

25. A method for detecting a target nucleic acid in claim 24, characterized in that step (d) irradiates light toward a direction that is not coated with metal of the retroreflective particles arranged in step (c).

26. A method for detecting a target nucleic acid comprising the following steps: (a) a step of hybridizing a trigger nucleic acid obtained from a sample containing a target nucleic acid and a sensor DNA of claim 1 comprising a sequence complementary to the trigger nucleic acid, and then treating the hybrid with a DNA polymerase to form a double-stranded nucleic acid; (b) a step of synthesizing a reporter protein from the formed double-stranded nucleic acid; (c) mixing the synthesized reporter protein with the retroreflective particle to which the first peptide is attached, and treating the substrate to which the second peptide is fixed, thereby arranging the retroreflective particle in a certain direction on the substrate; and (d) A step of detecting a retroreflective signal generated from a retroreflective particle by irradiating light.

27. A method for detecting a target nucleic acid according to claim 21 or 26, characterized in that the target nucleic acid is a nucleic acid derived from a food poisoning pathogen.

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