KR20220028919A - Kit for detecting biomarker using toehold switch system and detection method thereof - Google Patents

Abstract

The present invention relates to a kit for detecting a biomarker using a toehold switch system and a detection method using the same, and more specifically, to a hairpin structure oligomer comprising a toehold region; cell-free reaction solution; and a kit for detecting CD3ε including a substrate and a method for detecting SARS-CoV-2. It has been confirmed that the detection kit according to the present invention can detect CD3ε mRNA, which is a diagnostic indicator of acute kidney transplant rejection, with excellent efficiency, by accurately detecting target mRNA in a simpler way and within a faster time than conventional kits. Accordingly, the kit may be used to diagnose acute kidney transplant rejection at an early stage, and this technology can be advantageously used to replace existing methods in the detection of SARS-CoV-2, which has a high spread rate and a high infection rate.

Description

Kit for detecting biomarker using toehold switch system and detection method thereof

The present invention relates to a kit for detecting a biomarker using a toehold switch system and a detection method using the same, and more particularly, to a hairpin structure oligomer including a toehold region; cell-free reaction solution; And it relates to a kit for detecting CD3ε comprising a substrate or a method for detecting SARS-CoV-2 using the same.

After kidney transplantation, early diagnosis of acute rejection in transplant recipients is very important. To this end, needle biopsy and PCR (quantitative PCR and droplet digital PCR) are widely used in clinical practice for the diagnosis of acute rejection, but these methods still have their own drawbacks in some aspects.

Meanwhile, since December 2019, the pandemic of the coronavirus (SARS-CoV-2) first discovered in Hubei, China has killed a huge number of people worldwide and still threatens millions more. A large-scale pre-infection diagnosis has proven to be one of the effective measures to prevent the spread of a pandemic. Although RT-PCR is considered as a standard diagnostic method with high accuracy and sensitivity, it has its own limitations that it requires a long waiting time, expensive equipment, time, and a lot of labor. Since SARS-CoV-2 has a high rate of spread and high infectivity, many research groups are developing an alternative detection method with faster, simpler and more accurate detection efficiency, which can play an important role in suppressing the spread of infection.

Toehold switch is an RNA hairpin structure having a ribosome biding site (RBS) and an initiation codon (AUG) in a loop and a bulge and a single-stranded toehold region ( Toehold region) is connected. The toehold region binds to a trigger RNA that stimulates the operation of the toehold switch, whereby when the hairpin structure is opened and the RBS sequence is exposed, the ribosome binds to the recognition sequence and is linked to the back of the hairpin structure The translation of beta-galactosidase (β-galactosidase, LacZ) is initiated (Cell, 2014, 159, 925-39).

In addition, by combining an isothermal RNA amplification method called nucleic acid sequence-based amplification (NASBA), it is possible to optimize sensitivity by reducing the detection limit of the toehold switch. NASBA, a sensitive isothermal transcription-based system, utilizes three enzymes (avian myeloblastosis virus reverse transcriptase, RNase H and T7 RNA polymerase) to amplify single-stranded RNA under isothermal conditions. Useful as a diagnostic tool.

Tohold switch, an alternative riboregulator developed by the Wyss Institute of Harvard University, detected the RNA of various pathogenic viruses such as Ebola virus and Zika virus, and reported the result of diagnosing infection. there has been The above results showed the high specificity and sensitivity of the toehold switch, demonstrating that the system can be further applied in the future. A characteristic of the toehold switch is that it is possible to construct a specific toehold switch for a number of different RNAs. Therefore, if a toehold switch is manufactured by applying a sequence specific to the RNA to be detected, it may be utilized in various fields.

In the previous invention, a toehold switch was applied in the detection of IP-10 mRNA. However, the probability of a false positive may be high for a diagnosis based on only one RNA. Accordingly, the present inventors applied a toehold switch system to prepare a toehold switch that can specifically detect CD3ε mRNA in addition to IP-10 mRNA, which is a diagnostic marker for acute renal transplantation rejection, and the toehold switch and It was confirmed that the RNAs can be detected with high specificity and sensitivity using the cell-free reaction solution platform, thereby completing the invention. Furthermore, a SARS-CoV-2 detection method using a toehold switch was designed.

Accordingly, an object of the present invention is to provide a kit and a detection method for detecting CD3ε, which is an indicator of kidney transplant rejection.

Another object of the present invention is to provide a method for detecting SARS-CoV-2 using a toehold switch.

However, the technical problem to be achieved by the present invention is not limited to the above-mentioned problems, and other problems not mentioned will be clearly understood by those skilled in the art from the following description.

In order to achieve the object of the present invention as described above, the present invention provides a toehold region, a double-stranded nucleic acid sequence 1, a loop sequence 1, a double-stranded nucleic acid sequence 2, a loop sequence 2, a linker sequence and an expression nucleic acid sequence hairpin structure oligomer comprising;

cell-free reaction solution; and

Provided is a kit for detecting CD3ε, including a substrate.

In one embodiment of the present invention, the toehold region may be complementary to CD3ε mRNA or SARS-CoV-2 mRNA.

In another embodiment of the present invention, the expression nucleic acid sequence may include a gene sequence encoding β-galactosidase (LacZ).

In another embodiment of the present invention, the hairpin structure oligomer for detecting CD3ε may be any one selected from the group consisting of nucleotide sequences of SEQ ID NOs: 1 to 8.

In another embodiment of the present invention, the hairpin structure oligomer for detecting SARS-CoV-2 may be any one selected from the group consisting of nucleotide sequences of SEQ ID NO: 9 to SEQ ID NO: 68.

In another embodiment of the present invention, the cell-free reaction solution may include amino acids, rNTPs, tRNAs, RNA polymerase, translation factors, aminoacyl-tRNA synthetase (aminoacyl-tRNA synthetase), and ribosomes.

In another embodiment of the present invention, the substrate may be chlorophenol red-β-D-galactopyranoside (CBRG).

In addition, the present invention provides a method for detecting CD3ε or SARS-CoV-2, comprising the steps of:

(a) reacting by adding a biological sample derived from a subject to the kit; and

(b) confirming the color change of the reaction solution due to the colorimetric reaction.

In one embodiment of the present invention, the biological sample may be selected from the group consisting of tissues, cells, whole blood, blood, saliva, sputum, cerebrospinal fluid, and urine.

In another embodiment of the present invention, before reacting the biological sample with the kit in step (a), the method further comprises performing a nucleic acid sequence-based amplification (NASBA) reaction using the sample. can do.

In another embodiment of the present invention, the reaction in step (a) may be made for 2 to 6 hours.

In another embodiment of the present invention, when the color of the reaction solution changes from yellow to purple, it may be determined that CD3ε mRNA is present.

It was confirmed that the detection kit according to the present invention can detect CD3ε mRNA, which is a diagnostic index of acute kidney transplantation, with excellent efficiency, because it is possible to accurately detect target mRNA in a simpler and faster time than conventional detection and diagnostic means. Acute transplant rejection can be diagnosed at an early stage, and this technology will be useful as an alternative to existing methods for the detection of SARS-CoV-2, which has a high rate of spread and infection.

Figure 1 shows the screening results for eight candidate groups (70, 71, 72, 109, 175, 176, 291, 297) of the CD3ε mRNA detection threshold switch, Figure 1a is 100 nM of each threshold switch and 1 μM trigger mRNA CD3ε is the result of measuring the change in absorbance over time while incubating it at 37 ° C for 240 minutes, Figure 1b is the result showing the LacZ production rate of each toehold switch as a fold change after incubation for 240 minutes , Figure 1c is a result showing the color change of the reaction solution through interaction with the CPRG substrate according to the LacZ generation of each toehold switch.
Figure 2 is the result of analyzing the specificity of the toehold switch, after incubating IP-10, CD3ε and 18S RNA at 37°C for 240 minutes with 100 nM of two types of toehold switches (175 and 176) and trigger RNAs, respectively. These are the results showing the change in absorbance fold according to the LacZ generation and the results showing the color change of the reaction solution through interaction with the CPRG substrate according to the LacZ generation.
Figure 3 is a result of analyzing the sensitivity of the toehold switch, the toehold switch (No. 175) and the trigger RNA CD3ε mRNA at various concentrations (1000, 100, 10, 1, 0.1, 0 nM) at 37 ℃ 240 minutes These are the results showing the change in absorbance fold according to LacZ generation after culturing for a while, and the results showing the color change of the reaction solution through interaction with the CPRG substrate according to LacZ generation.
Figure 4 is the result of analyzing whether nucleic acid sequence-based amplification (NASBA) improves the detection sensitivity of the threshold switch for the trigger RNA, respectively, CD3ε and IP-10 trigger mRNA at various concentrations (100, 10, 1, 0.1 pM) with the toehold switch and incubation at 37°C for 240 minutes, the absorbance fold change according to LacZ generation was measured, and it was confirmed that the detection sensitivity of the toehold switch was improved through binding with NASBA.

The present inventors applied a toehold switch system to fabricate a toehold switch capable of specifically detecting CD3ε mRNA and SARS-CoV-2 RNA, which are diagnostic markers of acute kidney transplantation rejection, respectively, with high accuracy Through the detection of , it was confirmed that it can be used for diagnosis of acute kidney transplant rejection and infection with the virus.

Accordingly, the present invention provides a hairpin structure oligomer comprising a toehold region, a double-stranded nucleic acid sequence 1, a loop sequence 1, a double-stranded nucleic acid sequence 2, a loop sequence 2, a linker sequence and an expression nucleic acid sequence;

cell-free reaction solution; and

Provided is a kit for detecting CD3ε, including a substrate.

In the present invention, the hairpin structure oligomer has a toehold region, a double-stranded nucleic acid sequence 1, a loop sequence 1, a double-stranded nucleic acid sequence 2, a loop sequence 2, a linker sequence, and a beta-galactosidase gene sequence. It has the same meaning as the term 'toe hold switch' used in the present invention. The number of bases of the oligomer sequence may be adjusted for detection of trigger RNA with optimal efficiency, and specifically, it may consist of 60 to 120 nucleotides, preferably 70 to 110, even more preferably 80 to 100 nucleotides. there is.

The “toehold region” is a region that binds to a target RNA to be detected, that is, a trigger RNA, and is composed of a sequence complementary to a partial region of the trigger RNA. In the present invention, the toehold region may bind to CD3ε mRNA or SARS-CoV-2 mRNA, and may preferably consist of 10 to 30 nucleotides, more preferably 15 to 25 nucleotides.

In the present invention, the SARS-CoV-2 mRNA may preferably be an mRNA of the SARS-CoV-2 E gene (gene ID: 43740570).

In the present invention, the "nucleic acid sequence 1" and "nucleic acid sequence 2" each consist of a double-stranded, and nucleic acid sequence 1 is 5 to 15 nucleotides, preferably 10 to 15, more preferably 11 to 13 , most preferably 12 nucleotides, nucleic acid sequence 2 is 1 to 10 nucleotides, preferably 2 to 8, more preferably 3 to 7, still more preferably 4 to 6, Most preferably, it may consist of 5 nucleotides, and the number of nucleotides may be appropriately adjusted for the preparation of the oligomer.

The loop sequence refers to a sequence that is designed so that no complementary sequence exists in the sequence and forms a ring-shaped structure when the nucleic acid sequences at both ends form a double strand. In the present invention, "loop 1" includes a nucleotide sequence encoding an initiation codon (AUG) at which translation starts, and "loop 2" includes a nucleotide sequence encoding a ribosome bidding site (RBS). include Loop 2 may consist of 5 to 15 nucleotides, preferably 10 to 15, more preferably 11 to 13, and most preferably 12 nucleotides, and the number of nucleotides in the loop 1 and loop 2 sequences is appropriately can be adjusted.

In the present invention, the linker sequence is 15 to 30 nucleotides, preferably 17 to 25, more preferably 19 to 23, still more preferably 20 to 22, most preferably 21 It may consist of nucleotides.

In the present invention, the expression nucleic acid sequence may include a gene sequence encoding beta-galactosidase (β-galactosidase; LacZ). The gene encoding "beta-galactosidase (LacZ)" of the present invention is a gene that is translated into a protein by binding of the toehold region and trigger RNA, and the beta-galactosidase protein and the substrate The presence of RNA can be confirmed through the reaction of

In a specific example, the present inventors synthesized various hairpin structure oligomers including a toehold region using CD3ε mRNA and SARS-CoV-2 mRNA of E gene, respectively, as templates. Effective oligomers were selected by analyzing the detection specificity, sensitivity, and defect value of trigger RNA, that is, CD3ε mRNA and SARS-CoV-2 E gene mRNA, respectively.

In the present invention, the hairpin structure oligomer for detecting CD3ε may be any one selected from the group consisting of nucleotide sequences of SEQ ID NO: 1 to SEQ ID NO: 8.

In the present invention, the hairpin structure oligomer for detecting SARS-CoV-2 may be any one selected from the group consisting of nucleotide sequences of SEQ ID NO: 9 to SEQ ID NO: 68.

In the present invention, the cell-free reaction solution contains components necessary for translation of the LacZ gene constituting the hairpin structure oligomer, for example, amino acids, rNTPs, tRNA, RNA polymerase, translation factor, aminoacyl-tRNA synthetase (aminoacyl-tRNA synthetase) and ribosomes, but is not limited thereto, and may include all necessary components.

In the present invention, the substrate is used to induce a change in the color of the reaction solution to a purple (purple) series by a product generated through interaction with a protein translated from the LacZ gene encoded in the hairpin structure oligomer according to the present invention. For this purpose, it may be preferably chlorophenol red-β-D-galactopyranoside (CBRG), but is not limited thereto.

As another aspect of the present invention, the present invention provides a method for detecting CD3ε mRNA comprising the following steps.

(a) reacting by adding a biological sample derived from a subject to the kit; and

(b) confirming the color change of the reaction solution due to the colorimetric reaction.

In the present invention, the biological sample may be selected from the group consisting of extracorporeally separated tissues, cells, whole blood, blood, saliva, sputum, cerebrospinal fluid, and urine, but is not limited thereto.

In the step (a) of the present invention, before reacting the biological sample with the kit, the method may further include performing a nucleic acid sequence-based amplification (NASBA) reaction using the sample.

In one embodiment of the present invention, as a result of reacting with the toehold switch system using the number of NASBA and the reactant using CD3ε and IP-10 mRNA, which are indicators of kidney transplant rejection, the detection sensitivity of the system is in the nM unit. It was confirmed that the pM unit concentration of trigger RNA could be detected, and thus the sensitivity was greatly improved (see Example 5).

The reaction in step (a) may be 2 hours to 6 hours, preferably 3 to 5 hours, more preferably 4 hours, which is a reaction between the expressed beta-galactosidase and the substrate. It includes the time until the product production rate reaches a plateau.

In the present invention, the color change of the reaction solution can be determined by visually observing or measuring the absorbance of chlorophenol red, which is a product of the substrate in the reaction solution. When the color change is observed with the naked eye, if it changes from yellow to purple (purple), it may be determined that CD3ε mRNA is present.

Hereinafter, preferred examples are presented to help the understanding of the present invention. However, the following examples are only provided for easier understanding of the present invention, and the contents of the present invention are not limited by the following examples.

[Example]

Example 1. Experimental materials and methods

1-1. In silico Toehold Switch Design

In the present invention, the toehold switch manufactured for the detection of CD3ε and SARS-CoV-2 was designed based on the design scheme disclosed in the conventionally known prior literature (Cell, 165, 1255-1266). Briefly, the threshold region of the switch was designed to bind to a contiguous 30-nt region of the template RNA (CD3ε, viral E gene). Libraries for the toehold portion were generated in 1-nt increments over the full-length template RNA sequence. Other parts of the switch applied the conserved regions disclosed in the preceding literature above. Optimized conserved regions are the top-stem (5 bp) and loop region (12 nt) with a 21 nt linker region.

Candidate threshold regions generated from the template sRNA were screened for specificity using BLAST. Next, it was checked whether the designed toe hold switch avoids the stop codon in the frame. Selected switches were evaluated for interaction with trigger RNA by calculating NUPACK analysis and utility modules. Simultaneous defect values and the minimum free energy (MFE) of the secondary structure of the toehold switch and the structure of the switch/trigger RNA complex were calculated.

1-2. Structure of toe hold switch

The DNA template of the toehold switch was synthesized by Macrogen by adding two overhangs for the latter ligation with the beta-galactosidase (β-galactosidase) coding gene (LacZ) and vector. The template was amplified by PCR and purified by gel extraction. The DNA fragment was ligated with the LacZ gene using Gibson Assembly. The PCR-amplified product was inserted into a T7-based expression plasmid (pET28) containing LacI and Kanamycin resistance genes using Gibson Assembly with a total overlapping region of 30 bp, followed by DH5α E. coli strain was introduced into the plasmid.

For optimized transcription, a linear DNA template of both trigger RNAs was constructed by PCR using a primer pair designated to add a T7 promoter sequence to the 5' end of the PCR product. The linear DNA sequence of the toehold switch was prepared by PCR using the following primer pair. FWD_T7 promoter (5'-TAATACGACTCACTATAGGG-3', SEQ ID NO: 69) and REV_T7 terminator (5'-CAAAAAACCCCTCAAGAC-3', SEQ ID NO: 70).

Next, trigger RNA and RNA-type toehold switches were synthesized from the corresponding DNA template using a T7 RNAP-based transcription kit (MEGAscript® Kit, Thermo Fisher). RNA was then purified using the MEGAclear™ kit (Thermo Fisher) according to the manufacturer's protocol. The concentration of RNA was checked using a NanoDrop One spectrophotometer (Thermo Fisher) and stored at -20°C.

1-3. Preparation of Cell-Free Platform Reactants

A cell-free reaction solution was prepared using the PURExpress In Vitro Protein Synthesis Kit (NEB, E6800S). Specifically, solution A contains tRNA and small molecules including amino acids and rNTPs, while solution B contains protein components such as T7 RNA polymerase, translation factor, aminoacyl-tRNA synthetase and energy regenerative enzyme and ribosomes. Included. The rest is RNA inhibitor (20 U/30 μL reaction, NEB, Murine), chlorophenol red-β-D-galactopyranoside (CBRG, 0.6 mg/ L), RNA of the toehold switch (100 nM) and trigger RNA CD3ε (1 μM). The colorimetric change was evaluated every 15 minutes until the absorbance reached a plateau by measuring at a wavelength of 570 nm, which is the maximum absorbance of the chlorophenol red product generated from the LacZ reaction in the cell-free reaction solution during incubation at 37°C.

1-4. Toehold switch screening

Toehold switches designed using reaction kinetics were screened. Absorbance measurement data of the cell-free reaction solution over time, measured at 15-minute intervals, were derived by measuring with a NanoDrop One spectrophotometer (Thermo Fisher) at a wavelength of 570 nm. The screening of the toehold switch was performed based on the result of the fold change of absorbance. Switches having a relatively large fold change and reaching the maximum absorbance in a short incubation time were selected.

1-5. Specificity and Sensitivity Analysis of Toehold Switches

In order to confirm the specificity and sensitivity of the toehold switch, a cell-free reaction solution was prepared according to the method of Examples 1-3, and the absorbance of the reaction solution was measured after incubation at a wavelength of 570 nm for 240 minutes.

The specificity of the toehold switch was evaluated using other RNAs, i.e., non-homologous trigger RNAs such as IP-10 mRNA and 18S rRNA. The RNA contained in the urine was synthesized from a DNA template according to the RNA synthesis protocol, and was added to the cell-free reaction solution at a concentration of 100 nM to evaluate the specificity of the toehold switch. The specificity of the toehold switch was evaluated by comparing the absorbance of the reaction solution using the trigger mRNA CD3ε with the absorbance of the reaction solution using IP-10 mRNA and 18S rRNA, respectively.

The sensitivity of the toehold switch was analyzed using the concentration of trigger RNA CD3ε in the range of 1000 nM to 0.1 nM. The absorbance of the reaction solution was compared with the absorbance of the reaction solution including only the toe hold switch.

1-6. NASBA for Optimization of Sensitivity

NASBA reactions were used to optimize both the IP-10 and CD3ε toehold switches. For the NASBA reaction, an RNA amplicon containing a threshold-complementarity region (136-nt region for IP-10, 137-nt region for CD3ε) was used as a template for NASBA primer design. Reaction buffer (Life Sciences Advanced Technologies, NECB-24), nucleotide mix (Life Sciences Advanced Technologies, NECN-24), RNase inhibitor (Roche), nuclease free water and trigger mRNA (IP-10 or CD3ε) under the manufacturer's protocol Mixed at 4°C, incubated at 65°C for 2 minutes, and then incubated at 41°C for 10 minutes. Next, enzyme mix (Life Sciences Advanced Technologies, NEC-1-24) was added to the reaction (final volume 5 mL) and the mixture was incubated at 41° C. for 2 hours. NASBA reactions were diluted 1:5 in water for use in cell-free reactions. IP-10 and CD3ε were used at concentrations of 100 pM, 10 pM, 1 pM and 0.1 pM to evaluate the efficiency of NASBA.

1-7. Measurement of fold change in absorbance

The fold change value of absorbance represents the difference in the color change rate, which means the production rate of LacZ in the induced reaction. In order to exclude the effect of the cell-free system and the substrate chlorophenol red-β-D-galactopyranoside on absorbance, a control reaction solution containing no toehold switch and trigger RNA was used as a blank. The fold change was calculated by dividing the absorbance of the reaction solution at each time point by the absorbance of the control reaction solution including only the toehold switch. Measurement error was calculated from the standard deviation of 3 replicates.

Example 2. Toehold switch design for detection of CD3ε mRNA in urine

1, the toehold switch is a synthetic riboswitch that includes a hairpin structure and a toehold region. When the hairpin structure is opened by complementary binding with a trigger RNA, the ribosome binds to the ribosome binding region. The transformation is initiated. Based on the DNA template of urine RNA CD3ε, a total of 302 candidate toehold switches were designed and fabricated. Then, by removing the switches containing 4 or more consecutive identical nucleotide sequence patterns (AAAA, TTTT, CCCC, GGGG), 145 threshold switches were selected, and the threshold switches and frames with high homology with other human gene sequences 47 switches were selected by removing the toehold switch with the stop codon inside. Finally, a total of eight toehold switches (70, 71, 72, 109, 175, 176, 291 and 297) having a low coupling value were selected to perform synthesis and efficiency verification.

Example 3. Toe hold switch in vitro screening

The detection ability of trigger mRNA CD3ε was compared using the eight toehold switches selected in Example 2 above. As a result of the experiment, as shown in FIG. 1a , the change in absorbance with respect to most of the toehold switches was found to be alleviated by slowing down the LacZ production rate after incubation for 240 minutes. In addition, only two of the eight toehold switches (175 and 176) showed a high rate of LacZ production through significant differences in the colorimetric change seen in Figure 1b compared to the control response (36-fold and 10-fold, respectively). In addition, as can be seen from the result of FIG. 1c, it was possible to distinguish the colorimetric change of the two switches with the naked eye.

Example 4. Confirmation of specificity and sensitivity of toehold switch

3 shows the results of specificity analysis for the two types of toehold switches selected above. As a result of measuring the absorbance of the cell-free reaction solution after incubation for 240 minutes using each of the trigger mRNA and the non-trigger RNA, the 175 toehold switch showed a significant difference in the fold change of the LacZ absorbance between the two RNAs ( 10-fold for IP-10 mRNA, 36-fold for CD3ε mRNA, and 4-fold for 18S rRNA). Toehold switch 176 also showed relatively high specificity for CD3ε mRNA compared to the other two RNAs, but the difference in absorbance was much lower than that of 175. Therefore, it can be seen that the 175 toehold switch is the most effective for the detection of trigger CD3ε mRNA.

Furthermore, it was attempted to confirm the detection sensitivity of the 175 toehold switch by using CD3ε mRNA in a concentration range of 1000 nM to 0.1 nM. As a result, as shown in FIG. 4, when CD3ε mRNA was used at 1000 and 100 nM concentrations, respectively, the fold change in absorbance value was the highest at 36-fold and 42-fold, respectively. At concentrations ranging from 10 nM to 0.1 nM, the absorbance fold change was less than 10, but much higher than that of the control response. In terms of the visible colorimetric change, the colorimetric change of the reaction at the concentrations of 1000 nM and 100 nM exhibited a vivid purple color that can be recognized with the naked eye. In the case of concentration range of 10 ~ 0.1 nM, compared to the results at 1000 nM and 100 nM, the colorimetric change was not evident, and it appeared as an orange color.

Example 5. Isothermal Amplification NASBA for Sensitivity Optimization

NASBA was performed in the trigger mRNA regions of IP-10 and CD3ε, corresponding to the respective mRNA regions of toehold switches 1 and 175. The NASBA reaction was run for 2 hours and the product was used in a cell-free platform-based toehold switch system. As a result, as shown in FIG. 5 , in the case of CD3ε, the fold change of the reaction solution using the trigger RNA amplified with NASBA was higher than when the non-NASBA trigger RNA was used, and the detection sensitivity was lowered to 10 pM. confirmed that. This shows that the sensitivity is improved compared to the sensitivity confirmed in Example 4. In addition, in the case of IP-10, it was confirmed that the detection limit of the NASBA amplification trigger mRNA IP-10 was reduced to a level of 10 pM. NASBA amplification with trigger mRNA IP-10 at 1 and 0.1 pM showed distinct differences in fold change in LacZ absorbance compared to that with non-NASBA trigger mRNA. The results of this experiment showed the possibility that NASBA could be performed in combination with a cell-free platform-based toehold switch system to optimize the sensitivity of the system for detecting low concentrations of trigger RNA.

Example 6. Application of Toehold Switch Design Approach for Designing Toehold Switches for SARS-CoV-2 Detection

The present inventors confirmed that the toehold switch design is widely applicable to the detection of various RNAs. Specifically, it was intended to fabricate a toehold switch for detecting SARS-CoV-2 using the above design method. For the detection of the virus, the sequence of the virus E gene in the SARS-CoV-2 genome (NC_045512.2) was converted to a toehold switch It was used as a template for the design. As a result, a total of 199 candidate toehold domains were designed based on the viral E gene. In the candidate group, 112 top domains were selected by removing the top domain comprising a sequence pattern of 4 or more consecutive identical nucleotides, such as AAAA, TTTT, CCCC, GGGG, and then specificity was tested using BLAST, At this time, there was no toehold domain to be removed. The design of the toehold switch was completed by adding the conserved region described in Example 1-2 above, and then the toehold switch that did not include a stop codon in the frame was selected, and as a result, 100 switches were selected. Defect values for the complex structure with the viral E gene were then calculated for the selected toehold switches. As a result, 60 toehold switches with low defect values (< 20 %) were derived. An efficient switch can be further selected by analyzing high dynamics, specificity, and sensitivity for the derived toehold switch.

The description of the present invention stated above is for illustration, and those of ordinary skill in the art to which the present invention pertains can understand that it can be easily modified into other specific forms without changing the technical spirit or essential features of the present invention. There will be. Therefore, it should be understood that the embodiments described above are illustrative in all respects and not restrictive.

<110> University-Industry Cooperation Group of Kyung Hee University <120> Kit for detecting biomarker using toehold switch system and detection method thereof <130> PD20-227 <160> 70 <170> KoPatentIn 3.0 <210> 1 <211> 91 <212> DNA <213> Artificial Sequence <220> <223> Toehold switches_CD3E_175 <400> 1 gacaggtgat cctcatcact gcctatgttt ggactttaga acagaggaga taaagatgaa 60 acataggcag aacctggcgg cagcgcaaaa g 91 <210> <212> DNA <211> Artificial Sequence <220> <223> Toehold switches_CD3E_176 <400> 2 tgacaggtga tcctcatcac tgcctatgtt ggactttaga acagaggaga taaagatgaa 60 cataggcagt aacctggcgg cagcgcaaaa g 91 <210> 3 <211e_213> 91 <212> DNA <223> Artificial Tohold switches <212> 400> 3 gtcaatatta ctgtggttcc agagatggag ggactttaga acagaggaga taaagatgct 60 ccatctctgg aacctggcgg cagcgcaaaa g 91 <210> 4 <211> 91 <212> DNA <213> Artificial Sequence <220> <223> Toehold switches_CD3 agatttaga attc t ggt aga agatttaga <400> 60 catctctgga a acctggcgg cagcgcaaaa g 91 <210> 5 <211> 91 <212> DNA <213> Artificial Sequence <220> <223> Toehold switches_CD3E_72 <400> 5 atgtcaatat tactgtggtt ccagagatgg ggactttaga acagaggaga taaagatgca <210 aacct ggcgg cag ggcc> 60 atctctggaa 211> 91 <212> DNA <213> Artificial Sequence <220> <223> Toehold switches_CD3E_109 <400> 6 tgttgccata gtatttcaga tccaggatac ggactttaga acagaggaga taaagatggt 60 atcctggatc aacctggcgg cagcgcaaaa g 91 <210> 7211> DNA <210> 7211 > Artificial Sequence <220> <223> Toehold switches_CD3E_291 <400> 7 gttctcacac actcttgccc tcaggtagag ggactttaga acagaggaga taaagatgct 60 ctacctgagg aacctggcgg cagcgcaaaa g 91 <210> 8 <211> < 91 <220> Artificial Sequence <220> DNA Toehold switches_CD3E_297 <400> 8 catgcagttc tcacacactc ttgccctcag ggactttaga acagaggaga taaagatgct 60 gagggcaaga aacctggcgg cagcgcaaaa g 91 <210> 9 <211> 91 <212> DNA <223> Artificial Sequence3 <220> 9 gtacctgtct cttccgaaac gaatgagtac ggactttaga acag aggaga taaagatggt 60 actcattcgt aacctggcgg cagcgcaaaa g 91 <210> 10 <211> 91 <212> DNA <213> Artificial Sequence <220> <223> Toehold switches_SARS-CoV-2_5 <400> 10 acgtacctagat ctctactagaccgaa acgaatgagt t acccattttaggt t aggactt cagcgcaaaa g 91 <210> 11 <211> 91 <212> DNA <213> Artificial Sequence <220> <223> Toehold switches_SARS-CoV-2_6 <400> 11 aacgtacctg tctcttccga aacgaatgag ggactttaga acagaggaga taaagatgct 60 cattcgtttc aacct gga g cag <210 aacct gga g 91 > 12 <211> 91 <212> DNA <213> Artificial Sequence <220> <223> Toehold switches_SARS-CoV-2_7 <400> 12 taacgtacct gtctcttccg aaacgaatga ggactttaga acagaggaga taaagatgtc 60 attcgtttcg aacctggcgg cagcgcaaa> 91 <212> DNA <213> Artificial Sequence <220> <223> Toehold switches_SARS-CoV-2_8 <400> 13 ttaacgtacc tgtctcttcc gaaacgaatg ggactttaga acagaggaga taaagatgca 60 ttcgtttcgg aacctggcgg cagcgca <aaa g 91 <213> Artificial Sequence <220> <223> Toehold switches_SARS-CoV-2 _9 <400> 14 attaacgtac ctgtctctttc cgaaacgaat ggactttaga acagaggaga taaagatgat 60 tcgtttcgga aacctggcgg cagcgcaaaa g 91 <210> 15 <211> 91_10> DNA <213> Artificial Sequence <220> <223> Toehold switches_SARS-CoV tattaacgta cctgtctctt ccgaaacgaa ggactttaga acagaggaga taaagatgtt 60 cgtttcggaa aacctggcgg cagcgcaaaa g 91 <210> 16 <211> 91 <212> DNA <213> cta ttaacgt ggacta ggta gt ac gt ggsars-CoV -2 taaagatgtc 60 gtttcggaag aacctggcgg cagcgcaaaa g 91 <210> 17 <211> 91 <212> DNA <213> Artificial Sequence <220> <223> Toehold switches_SARS-CoV-2_12 <400> 17 actattaacg taaacctgtgtagactc taggtgaaacg agg c g 91 <210> 18 <211> 91 <212> DNA <213> Artificial Sequence <220> <223> Toehold switches_SARS-CoV-2_13 <400> 18 aactattaac gtacctgtct cttccgaaac ggactttaga acagaggaga taaaagatggt 60 210 ttcggaagag aacctggcgg cagc 19 <211> 91 <212> DNA < 213> Artificial Sequence <220> <223> Toehold switches_SARS-CoV-2_14 <400> 19 taactattaa cgtacctgtc tcttccgaaa ggactttaga acagaggaga taaagatgtt 60 tcggaagaga aacctggcgg cagcgcaaaa g 91 <210> 20 <211> Artificial Sequence <210> 20 <211 220> <223> Toehold switches_SARS-CoV-2_15 <400> 20 ttaactatta acgtacctgt ctcttccgaa ggactttaga acagaggaga taaagatgtt 60 cggaagagac aacctggcgg cagcgcaaaa g 91 <210> 21 <211> 91 <212> DNA <213> Artificial Sequence> 223 Toehold switches_SARS-CoV-2_16 <400> 21 attaactatt aacgtacctg tctcttccga ggactttaga acagaggaga taaagatgtc 60 ggaagagaca aacctggcgg cagcgcaaaa g 91 <210> 22 <211> 91 <212> DNA <223> Artificial Sequence <220> Toehold switches_SA 2_17 <400> 22 tattaactat taacgtacct gtctcttccg ggactttaga acagaggaga taaagatgcg 60 gaagagacag aacctggcgg cagcgcaaaa g 91 <210> 23 <211> 91 <212> DNA <213> Artificial Sequence <220> <223> Toehold switches_18RS-CoV-2_18RS ctattaacta ttaacgtacc tgtctcttcc ggactttaga acagaggaga taaagatggg 60 aa gagacagg aacctggcgg cagcgcaaaa g 91 <210> 24 <211> 91 <212> DNA <213> Artificial Sequence <220> <223> Toehold switches_SARS-CoV-2_19 <400> 24 gctattaact attaacgtac ctgtctcttc ggactttaga acagaggaga taaagatgga 60 agagacaggt aacctggcgg cagcgcaaaa g 91 <210> 25 <211> 91 <212> DNA <213> Artificial Sequence <220> <223> Toehold switches_SARS-CoV-2_accttt actattact gg aga accttag <400 taaagatgag 60 agacaggtac aacctggcgg cagcgcaaaa g 91 <210> 26 <211> 91 <212> DNA <213> Artificial Sequence <220> <223> Toehold switches_SARS-CoVaca-2_22 <400> 26 tacggctatta cagtaacg agatacctgtctga actagaggaga taatta cagtaacg agatacctgtctc g 91 <210> 27 <211> 91 <212> DNA <213> Artificial Sequence <220> <223> Toehold switches_SARS-CoV-2_23 <400> 27 gtacgctatt aactattaac gtacctgtct ggactttaga acagaggaga taaagatgag 60 acaggtacgt aacctggcgg cagcgcaa> 28 <211> 91 <212> DNA <213> Artificial Sequence <220> <223> Toehold switches_SARS-CoV-2_24 <400> 28 agtacgctat taactattaa cgtacctgtc ggactttaga acagaggaga taaagatgga 210 caggtacgtt aacctggcgg cagcgcaaaa g 91 <212> DNA <213> Artific ial Sequence <220> <223> Toehold switches_SARS-CoV-2_25 <400> 29 aagtacgcta ttaactatta acgtacctgt ggactttaga acagaggaga taaagatgac 60 aggtacgtta aacctggcgg cagcgcaaaa g 91 <210> 30 <211> < 91 <212 Artificial> DNA <211> <212 <223> Toehold switches_SARS-CoV-2_27 <400> 30 agaagtacgc tattaactat taacgtacct ggactttaga acagaggaga taaagatgag 60 gtacgttaat aacctggcgg cagcgcaaaa g 91 <210> 31 <211> 91 <212> DNA <213> Artificial Sequence Toehold switches -CoV-2_28 <400> 31 aagaagtacg ctattaacta ttaacgtacc ggactttaga acagaggaga taaagatggg 60 tacgttaata aacctggcgg cagcgcaaaa g 91 <210> 32 <211> 91 <212> DNA <213> Artificial Sequence <220> <223> Toehold switches_68 400> 32 tggctagtgt aactagcaag aataccacga ggactttaga acagaggaga taaagatgtc 60 gtggtattct aacctggcgg cagcgcaaaa g 91 <210> 33 <211> 91 <212> DNA <213> Artificial Sequence <220> <223> Toehold switches_69 <400> Toehold switches_69 <400> gaataccacg ggactttaga acagaggaga taaagatgcg 60 tggtattctt aac ctggcgg cagcgcaaaa g 91 <210> 34 <211> 91 <212> DNA <213> Artificial Sequence <220> <223> Toehold switches_SARS-CoV-2_70 <400> 34 gatggctagt gtaactagca agaataccac ggactttaga acagaggaga taaagatgg cttg aacca gag cttg aacctatt 91 <220> <223> Toehold switches_SARS-CoV-2_70 210> 35 <211> 91 <212> DNA <213> Artificial Sequence <220> <223> Toehold switches_SARS-CoV-2_76 <400> 35 agtaaggatg gctagtgtaa ctagcaagaa ggactttaga acagaggaga taaagatgtt 60 cttgctagtt aacctcaaaa g 91 > 91 <212> DNA <213> Artificial Sequence <220> <223> Toehold switches_SARS-CoV-2_78 <400> 36 gcagtaagga tggctagtgt aactagcaag ggactttaga acagaggaga taaagatgct 60 tgctagttac aacctggcgg cagcgcaaaa < 91 <210> 37211 <210> 37211 DNA <213> Artificial Sequence <220> <223> Toehold switches_SARS-CoV-2_79 <400> 37 cgcagtaagg atggctagtg taactagcaa ggactttaga acagaggaga taaagatgtt 60 gctagttaca aacctggcgg cagcgcaaaa g 91 <210> 38 <211> Artificial Sequence <220> <223> Toehold switches_SARS-CoV-2_84 <400> 38 cgaagcgcag taaggat ggc tagtgtaact ggactttaga acagaggaga taaagatgag 60 ttacactagc aacctggcgg cagcgcaaaa g 91 <210> 39 <211> 91 <212> DNA <213> Artificial Sequence <220> <223> Toehold switches_SARS-CoV-2_87 <400> 39 agtaggaga ttaga tagctagt agaagc tagctagt g 60 cactagccat aacctggcgg cagcgcaaaa g 91 <210> 40 <211> 91 <212> DNA <213> Artificial Sequence <220> <223> Toehold switches_SARS-CoV-2_88 <400> 40 caatcgaagc gcagtaagga tggctagtgt ggactttaga aaccaggagga 60 g 91 <210> 41 <211> 91 <212> DNA <213> Artificial Sequence <220> <223> Toehold switches_SARS-CoV-2_91 <400> 41 acacaatcga agcgcagtaa ggatggctag ggactttaga acagaggaga taaaagatgct210 60 agccatcctt aacctggcgg <400> 42 <211> 91 <212> DNA <213> Artificial Sequence <220> <223> Toehold switches_SARS-CoV-2_95 <400> 42 acgcacacaa tcgaagcgca gtaaggatgg ggactttaga acagaggaga taaagatgcc 60 atccttactg aacctggcgg cagcgcaaaa g 91 212> DNA <213> Artificial Sequence <220> <223> Toehold switches_SARS-CoV-2_98 <400> 43 agtacgcaca caatcgaagc gcagtaagga ggactttaga acagaggaga taaagatgtc 60 cttactgcgc aacctggcgg cagcgcaaaa g 91 <210> 44 <211> 91 <212> DNA <223> Artificial Toehold switches <212> DNA <223> Toehold switches_SARS-CoV-2_98 <400> -CoV-2_103 <400> 44 gcagcagtac gcacacaatc gaagcgcagt ggactttaga acagaggaga taaagatgac 60 tgcgcttcga aacctggcgg cagcgcaaaa g 91 <210> 45 <211> 91 <105> DNA <213> Artificial Sequence <220> <223> 400> 45 ttgcagcagt acgcacacaa tcgaagcgca ggactttaga acagaggaga taaagatgtg 60 cgcttcgatt aacctggcgg cagcgcaaaa g 91 <210> 46 <211> 91 <212> DNA <213> Artificial Sequence <220> <223> Toeatt switches <220> <223> Toeatt switches gcacacaatc ggactttaga acagaggaga taaagatgga 60 ttgtgtgcgt aacctggcgg cagcgcaaaa g 91 <210> 47 <211> 91 <212> DNA <213> Artificial Sequence <220> <223> Toehold switches_SARS-CoV-2_114 <400> tgagaagacaatat cag tga tgagaagacaatat 60 tgtgtgcgta aacctggcgg cagcgc aaaa g 91 <210> 48 <211> 91 <212> DNA <213> Artificial Sequence <220> <223> Toehold switches_SARS-CoV-2_115 <400> 48 gttaacaata ttgcagcagt acgcacacaa ggactttaga acagaggaga taaagatgtt 60 gtgtgcgtac 210 aacctgga > 49 <211> 91 <212> DNA <213> Artificial Sequence <220> <223> Toehold switches_SARS-CoV-2_116 <400> 49 cgttaacaat attgcagcag tacgcacaca ggactttaga acagaggaga taaagatgtg210 60 tgtgcgtact aacctggcgg cagcgcaaaa g <91 <2> 50211> 91 <212> DNA <213> Artificial Sequence <220> <223> Toehold switches_SARS-CoV-2_117 <400> 50 acgttaacaa tattgcagca gtacgcacac ggactttaga acagaggaga taaagatggt 60 gtgcgtactg aacctggcgg cagcggcaaaa g 91 <213> Artificial Sequence <220> <223> Toehold switches_SARS-CoV-2_118 <400> 51 cacgttaaca atattgcagc agtacgcaca ggactttaga acagaggaga taaagatgtg 60 tgcgtactgc aacctggcgg cagcgcaaaa g 91 <210> <212> DNA <211> Artificial Sequence <220> <223> Toehold switches_SARS-CoV-2_119 <400> 52 tcacgttaac aatattgcag cagta cgcac ggactttaga acagaggaga taaagatggt 60 gcgtactgct aacctggcgg cagcgcaaaa g 91 <210> 53 <211> 91 <212> DNA <213> Artificial Sequence <220> <223> Toehold switches_SARS-CoV-2_120 <400> 53 ctcacgttaga gcagtg caat att taca aga gcagtg caat cgtactgctg aacctggcgg cagcgcaaaa g 91 <210> 54 <211> 91 <212> DNA <213> Artificial Sequence <220> <223> Toehold switches_SARS-CoV-2_121 <400> 54 actcacgtta acaatattgc agcagtacgc ggactttaga cagtggc acc 60 taa g <210> 55 <211> 91 <212> DNA <213> Artificial Sequence <220> <223> Toehold switches_SARS-CoV-2_122 <400> 55 gactcacgtt aacaatattg cagcagtacg ggactttaga acagaggaga taaagatgcg 60 tactgctgca aacctggcgg cagcgcaaa <210> 211> 91 <212> DNA <213> Artificial Sequence <220> <223> Toehold switches_SARS-CoV-2_123 <400> 56 agactcacgt taacaatatt gcagcagtac ggactttaga acagaggaga taaagatggt 60 actgctgcaa aacctggcgg cagcgcaaaa g 91 <210> 57211 <210> 57211 > DNA <213> Artificial Sequence <220> <223 > Toehold switches_SARS-CoV-2_124 <400> 57 aagactcacg ttaacaatat tgcagcagta ggactttaga acagaggaga taaagatgta 60 ctgctgcaat aacctggcgg cagcgcaaaa g 91 <210> 58 <RS211>V 91 <212> DNA <213> Artificial Sequence Toehold switches_SARS-CoV-2_124 <213> -2_125 <400> 58 caagactcac gttaacaata ttgcagcagt ggactttaga acagaggaga taaagatgac 60 tgctgcaata aacctggcgg cagcgcaaaa g 91 <210> 59 <211> 91 <212> DNA <213> Artificial Sequence <220> <223> Toehold switches <220> <223> Toehold switches 59 acaagactca cgttaacaat attgcagcag ggactttaga acagaggaga taaagatgct 60 gctgcaatat aacctggcgg cagcgcaaaa g 91 <210> 60 <211> 91 <212> DNA <213> Artificial Sequence <220> <223> Toehold switches_SARS-CoV-2_127 acagaggaga taaagatgtg 60 ctgcaatatt aacctggcgg cagcgcaaaa g 91 <210> 61 <211> 91 <212> DNA <213> Artificial Sequence <220> <223> Toehold switches_SARS-CoV-2_189 <400> 61 agatcaggaaga agactagactagaaaga atttcagattt t cagcgcaaaa g 91 <210> 62 <211> 91 <212> DNA <213> Artificial Sequence <220> <223> Toehold switches_SARS-CoV-2_190 <400> 62 aagatcagga actctagaag aattcagatt ggactttaga acagaggaga taaagatgaa 60 tctgaattct aacctggcgg cagcgcaaaa g 91 <210> 63 <211> 91 <212> DNA <213> Artificial Sequence <220> <223> Toehold switches_SARS-CoV-2_191 <400> 63 gaagatcagg aactctagaa gaattcagat ggactttaga acagaggaga taaagatgat 60 ctgaattctt aacctggcgg cagcgca <aaa g 91 <212> DNA <213> Artificial Sequence <220> <223> Toehold switches_SARS-CoV-2_192 <400> 64 agaagatcag gaactctaga agaattcaga ggactttaga acagaggaga taaagatgtc 60 tgaattcttc aacctggcgg cagcgcaaaa g 91 <210> 65 <211> 213> Artificial Sequence <220> <223> Toehold switches_SARS-CoV-2_193 <400> 65 cagaagatca ggaactctag aagaattcag ggactttaga acagaggaga taaagatgct 60 gaattcttct aacctggcgg cagcgcaaaa g 91 <210> 66 <211> < 91 <212> Artificial DNA 220> <223> Toehold switches_SARS-CoV-2_196 <400> 66 gaccagaaga tcaggaactc tagaagaat t ggactttaga acagaggaga taaagatgaa 60 ttcttctaga aacctggcgg cagcgcaaaa g 91 <210> 67 <211> 91 <212> DNA <213> Artificial Sequence <220> <223> Toehold switches_SARS-CoV-2_197 ac <400> 67 agact tagat atcagga ttaa agacctagaag atcagga tcttctagag aacctggcgg cagcgcaaaa g 91 <210> 68 <211> 91 <212> DNA <213> Artificial Sequence <220> <223> Toehold switches_SARS-CoV-2_199 <400> 68 ttagaccaga agatcaggaa 91ctagaaga ggactagat cagt accagatggaga taa gag <210> 69 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> FWD_T7_promoter <400> 69 taatacgact cactataggg 20 <210> 70 <211> 18 <212> DNA <213> Artificial Sequence <220 > <223> REV_T7_terminator<400> 70 caaaaaaccc ctcaagac 18

Claims (12)

a hairpin structure oligomer comprising a toehold region, a double-stranded nucleic acid sequence 1, a loop sequence 1, a double-stranded nucleic acid sequence 2, a loop sequence 2, a linker sequence, and an expression nucleic acid sequence;
cell-free reaction solution; and
A kit for the detection of CD3ε or SARS-CoV-2 comprising a substrate.
The method of claim 1,
The toehold region is characterized in that complementary binding to CD3ε mRNA or SARS-CoV-2 mRNA, the kit.
The method of claim 1,
The expression nucleic acid sequence is beta-galactosidase (β-galactosidase; LacZ) characterized in that it comprises a gene sequence encoding the kit.
The method of claim 1,
The kit, characterized in that the hairpin structure oligomer for detecting CD3ε is any one selected from the group consisting of nucleotide sequences of SEQ ID NO: 1 to SEQ ID NO: 8.
The method of claim 1,
The kit, characterized in that the hairpin structure oligomer for detecting SARS-CoV-2 is any one selected from the group consisting of nucleotide sequences of SEQ ID NO: 9 to SEQ ID NO: 68.
The method of claim 1,
The cell-free reaction solution comprises amino acids, rNTPs, tRNA, RNA polymerase, translation factors, aminoacyl-tRNA synthetase (aminoacyl-tRNA synthetase) and ribosomes, the kit.
The method of claim 1,
The kit, characterized in that the substrate is chlorophenol red-β-D-galactopyranoside (CBRG).
A method for detecting CD3ε or SARS-CoV-2, comprising the steps of:
(a) reacting by adding a biological sample derived from the subject to the kit of claim 1; and
(b) confirming the color change of the reaction solution due to the colorimetric reaction.
9. The method of claim 8,
The biological sample is a detection method, characterized in that selected from the group consisting of tissue, cells, whole blood, blood, saliva, sputum, cerebrospinal fluid and urine.
9. The method of claim 8,
Before reacting the biological sample with the kit in step (a), the detection method further comprising the step of performing a nucleic acid sequence-based amplification (NASBA) reaction using the sample .
9. The method of claim 8,
The reaction in step (a) is characterized in that it is made for 2 to 6 hours, the detection method.
9. The method of claim 8,
In step (b), when the color of the reaction solution changes from yellow to purple, it is characterized in that it is determined that the mRNA of CD3ε or SARS-CoV-2 is present.

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