KR20160054295A - DNA Aptamer Specifically Binding to Surface of Living Cell of Vibrio fischeri and Uses Thereof - Google Patents
DNA Aptamer Specifically Binding to Surface of Living Cell of Vibrio fischeri and Uses Thereof Download PDFInfo
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Abstract
The present invention relates to a DNA aptamer which specifically binds to the surface of a Vibrio fischeri live cell and its use. The present invention allows DNA aptamers to bind to the surface of Vibrio fischeri live cells in a high specificity. By using the DNA aptamer of the present invention, it is possible to detect and confirm the presence of Vibrio ficus-producing bacteria in the sample. Use of a DNA aptamer-antimicrobial substance complex in which an antimicrobial substance is conjugated to the DNA aptamer of the present invention can inhibit the growth of Vibrio ficus-indica and effectively inhibit the biofilm formed by the bacterium.
Description
The present invention relates to a DNA aptamer which specifically binds to the surface of a V. vulnificus and a use thereof.
Vibrio fischeri is a strain that grows on the light emitting organs of deep-sea squid among the Vibrio genus. It is a tuberous anaerobic gram-negative bacterium, which is motile and has one or more flagella. Vibrio fischeri is one of several strains of marine ecosystems and forms a biofilm as a strategy to increase viability. It is recognized that resistance to multiple stresses is significantly increased compared to non-species, thus becoming an essential step in survival strategy . However, when the biofilm is formed, there is a problem that the resistance of the therapeutic agent and the antibiotic against Vibrio ficusi is increased about 500 times as compared with that in the case of swimming. Therefore, biofilm formation has many adverse effects on the economy and industry even if it is a natural phenomenon as a means of survival strategy of an organism. Formation of biofilm has been found to account for 80% of the adverse effects of implant surgery.
Conventional methods for controlling the production of biofilm have been attempted based on bacterial killing by antibiotics or antifouling agents, but the indiscriminate use of antibiotics in strains with increased resistance may lead to mutations into antibiotic resistant strains, It is very limited. In addition, because antifouling agents are non-selective biotoxins that are toxic to other organisms, the use of antifouling agents causes not only great damage to environmental ecosystems but also serious economic damage to recover them. The risk of antifouling has been confirmed by the fact that TBT (tributyl tin), a chemical fouling agent most commonly used in ships and aquaculture since the 1970s, causes a variety of phenomena such as male genitalia in females of various marine organisms, It has been pointed out that it is an environmental hormone that seriously threatens the environment. TBT can induce sexual mutation even at concentrations below 1 ppt and there is continuing discovery of TBT contamination in the southern coast. Therefore, in developed countries, since 1982, the concentration of TBT used as an antifouling agent on large ships has been regulated, and it has been banned for small ships operating on the coast. In Korea, TBT regulations have been introduced since 2005, and substances such as copper sulfate have been used as an alternative material. However, TBT has been designated as a contaminant, and it is necessary to develop a substitute for an antifouling agent such as TBT.
Aptamers are composed of oligomers of short length and form their own various three-dimensional structures. These oligomer structure libraries are capable of structurally binding with various target substances, and among them, screening for aptamers specific to the target substance Respectively. The selected aptamers have the advantage of being able to obtain sequence through sequence analysis, mass production in a short time and low cost by using chemical synthesis technique, and continuous production of aptamer having the same ability once production and production. In addition, it is composed of DNA and can provide more stability through additional chemical substitution process. It is very stable in surrounding pH and temperature, and attaches compounds such as biotin, And it can be used in various fields such as medicine.
Numerous papers and patent documents are referenced and cited throughout this specification. The disclosures of the cited papers and patent documents are incorporated herein by reference in their entirety to better understand the state of the art to which the present invention pertains and the content of the present invention.
The present inventors have made efforts to develop a DNA aptamer that can be used for various purposes such as detection and growth inhibition of this bacterium by specifically binding to a viable state of Vibrio fischeri . As a result, we succeeded in synthesizing and screening DNA aptamers capable of binding specifically to the viable surface of Vibrio ficusi. Using this DNA aptamer, we developed a rapid kit platform that can easily detect Vibrio ficusii The present inventors completed the present invention by experimentally confirming the possibility of effectively blocking the biofilm produced by Vibrio fischeri by inhibiting the growth of Vibrio fischeri.
Accordingly, an object of the present invention is to provide a DNA aptamer which specifically binds to the surface of a live bacterium of Vibrio fischeri .
Another object of the present invention is to provide a composition and a kit for detecting Vibrio fischeri live cells comprising the DNA aptamer as an active ingredient.
It is still another object of the present invention to provide a method for detecting Vibrio fischeri live cells.
The objects and advantages of the present invention will become more apparent from the following detailed description of the invention, claims and drawings.
According to one aspect of the present invention, there is provided a DNA aptamer that specifically binds to the surface of a live bacterium of Vibrio fischeri .
According to one embodiment of the present invention, the DNA aptamer of the present invention is an oligonucleotide having a nucleotide sequence having 90% or more identity with the nucleotide sequence set forth in any one of SEQ ID NOS: 1 to 13.
According to another embodiment of the present invention, the DNA aptamer of the present invention is an oligonucleotide having a nucleotide sequence as set forth in any one of SEQ ID NOS: 1 to 13.
According to another embodiment of the present invention, the DNA aptamer of the present invention is chemically modified, modified or bound to its 5 'terminal or 3' terminal, and preferably biotin, Cy3, Cy5, fulorescein, or an oligonucleotide to which a radioactive substance is bound.
According to another aspect of the present invention, there is provided a composition for detecting Vibrio fischeri live cells comprising the DNA aptamer as an active ingredient.
According to another aspect of the present invention, there is provided a kit for detecting Vibrio fischeri live cells comprising the DNA aptamer as an active ingredient.
According to one embodiment of the present invention, the kit of the present invention is a chip in which DNA aptamers are immobilized on a chip.
According to another embodiment of the present invention, the kit of the present invention is in the form of a microarray in which DNA aptamers are immobilized on a chip.
According to another aspect of the present invention, the present invention provides a method of detecting Vibrio fischeri live bacteria comprising the steps of: (a) detecting the presence of Vibrio fischeri Contacting the sample with the DNA aptamer; And (b) identifying a Vibrio fischeri live cell coupled to the DNA aptamer.
According to another aspect of the present invention, the present invention provides a DNA-DNA polymerase comprising (i) the DNA aptamer; And (ii) an antimicrobial substance conjugated to the DNA aptamer through a linker.
According to another aspect of the invention there is provided the aptamer-of phase V. Fisher Lee (Vibrio fischeri) comprising a contacting a sample expected to contain the antibacterial substance complex a Vibrio Fisher Lee (Vibrio fischeri) live cells Thereby providing a method of inhibiting growth.
Hereinafter, the present invention will be described in more detail.
Vibrio Fishery ( Vibrio fischeri ) DNA aptamer binding to live cell surface
The present invention relates to a DNA aptamer that specifically binds to the surface of a live bacterium of Vibrio fischeri .
As used herein, the term " DNA aptamer " refers to a DNA nucleic acid molecule capable of binding with specificity and specificity to a specific target molecule. The term " DNA aptamer " can be used interchangeably as a substantially equivalent meaning to " DNA oligonucleotide ".
The term " oligonucleotide " as used herein generally refers to a nucleotide polymer having less than about 200 lengths, including DNA and RNA, preferably DNA nucleotide molecules. The nucleotides may be any substrate that can be introduced into the polymer by deoxyribonucleotides, ribonucleotides, modified nucleotides or bases and / or their analogs, or by DNA or RNA polymerases or by synthetic reactions. If a modification to the nucleotide structure is present, such modification may be added before or after the synthesis of the oligonucleotide polymer. The nucleotide sequence may be terminated by a non-nucleotide component. Oligonucleotides can bind label substances such as fluorescent materials after synthesis and can be chemically modified or modified to improve stability.
In the DNA aptamer of the present invention, for example, a fluorescent substance such as biotin, an amine group, a thiol group, a radioactive substance, Cy3, Cy5, or fluorescein may be bonded or introduced at the 5'- or 3'- . In order to improve the stability and efficiency of the DNA aptamer, the hydrogen located at the
The DNA aptamers of the present invention are typically obtained by in vitro selection for binding of target molecules. Methods for selecting an aptamer that specifically binds to a target molecule are known in the art. For example, organic molecules, nucleotides, amino acids, polypeptides, marker molecules on a cell surface, ions, metals, salts, polysaccharides can be suitable target molecules for separating aptamers that can specifically bind to each ligand .
Selection of an aptamer can utilize in vivo or in vitro selection techniques known in the SELEX (Systematic Evolution of Ligands by Exponential Enrichment) method (Ellington et al., Nature 346, 818-22, 1990; and Tuerk et al. , Science 249, 505-10, 1990). The term " SELEX method " as used herein means a method of extracting a DNA binding sequence of a molecule by selectively amplifying a DNA having a high binding force to a specific molecule from a set of arbitrarily synthesized DNAs (Louis et al. Nature 355, 564-566). Specific methods for selection and preparation of DNA aptamers are described in U.S. Patent 5,582,981, WO 00/20040, U.S. Patent 5,270,163, Lorsch and Szostak, Biochemistry, 33: 973 (1994), Mannironi et al., Biochemistry 36: , Blind, Proc. Natl. Acad. Sci. USA 96: 3606-3610 (1999), Huizenga and Szostak, Biochemistry, 34: 656-665 (1995), WO 99/54506, WO 99/27133, WO 97/42317 and U.S. Patent 5,756,291, Are incorporated herein by reference.
The DNA aptamer of the present invention is preferably an oligonucleotide having the nucleotide sequence set forth in any one of SEQ ID NOS: 1 to 13.
On the other hand, the DNA aptamer having the nucleotide sequence of any one of SEQ ID NOS: 1 to 13 of the present invention is presumed to form a specific secondary structure.
The DNA aptamer of the present invention is an oligonucleotide having a nucleotide sequence that exhibits substantial identity with any one of the nucleotide sequences shown in SEQ ID NOS: 1 to 13, while retaining the property of specifically binding to the surface of Vibrio ficus- .
The above-mentioned substantial identity can be determined by aligning the nucleotide sequence of the present invention with any other sequence as much as possible and using an algorithm commonly used in the art (Smith and Waterman, Adv. Appl. Math. 2: 482 (1981) Hewgins and Sharp, Gene 73: 237-44 (1988); < RTI ID = 0.0 > Higgins and Sharp, CABIOS 5: 151-3 (1989); Corpet et al., Nuc Acids Res. 16: 10881-90 (1988); Huang et al., Comp. (At least 90% identity, more preferably at least 95% identity, more preferably at least 95% identity) to the amino acid sequence of SEQ ID NO: Identity, most preferably at least 98% identity to the nucleotide sequence of SEQ ID NO: 1.
Screening of Vibrio Fischeri surface-bound DNA aptamers by Live Cell SELEX technique
DNA aptamers that specifically bind to the Vibrio ficus live cell surface of the present invention are selected by the Cell-SELEX method.
According to a specific embodiment of the present invention, the DNA aptamer of the present invention is selected through the following steps: (i) amplifying a DNA aptamer through a PCR (Polymerase Chain Reaction) technique and producing a ssDNA aptamer step; (Ii) culturing and washing Vibrio fischeri; (Iii) A step of selecting a DNA aptamer that specifically binds to the surface of Vibrio ficus live cells through a Live Cell SELEX technique using Vibrio ficus.
(I) amplifying a DNA aptamer using a PCR method and preparing a ssDNA aptamer:
First, a random dsDNA library is amplified using PCR. Asymmetric PCR is performed to amplify only the ssDNA in the amplified Landon dsDNA library. Asymmetric PCR is a method of obtaining ssDNA by performing PCR using a forward primer and a reverse primer at a ratio of 10: 1, for example, 10 μl of a forward primer and 1 μl of a reverse primer at the same concentration (25 μM). For example, when performing PCR, ssDNA aptamer is selected by, for example, attaching biotin to a reverse primer to amplify dsDNA and treating the amplified product with streptavidin to form a biotin-streptavidin complex, And selectively removing only the ssDNA aptamer. The ssDNA aptamers prepared are denatured by heating ssDNA for use in the SELEX method, and then slowly cooled at room temperature to form a three-dimensional structure.
(Ii) culturing and washing the Vibrio fischeri:
To screen for DNA aptamers that bind to viable surface of V. vulnificus, cultivate V. vulgaris. To maintain viable conditions, Vibrio ficus strains were grown on LBS medium [
(Iii) selecting a DNA aptamer that specifically binds to the surface of the viroid of the viroid by means of the Live Cell SELEX technique using the cultured viable viroid cells;
After the prepared Vibrio Fischeri and SsDNA aptamer are contacted and reacted with each other, the unbound ssDNA aptamer is washed and removed, and only the specifically binding ssDNA is eluted. At this stage, it may include a negative SELEX step for removing ssDNA binding to other bacteria, for example Gram-negative bacteria such as Escherichia coli and Gram-positive bacteria, Bacillus bacteria.
In the method of the present invention, in order to select the optimal SELEX round in which the DNA aptamer that specifically binds to the Vibrio ficus live cell surface is eluted to the maximum, a method of quantitating the ssDNA concentration using the nano-drop of the eluted ssDNA can be used have.
According to a specific embodiment of the present invention, in order to select a DNA aptamer exhibiting an optimum binding force with Vibrio ficusi during an optimal SELEX round in the method of the present invention, the optimal aptamer sequence . After SELEX was prepared with the same concentration of ssDNA, the concentration of eluted aptamers was measured by nano-drop method using nano-drop method to select optimal Vibrio Fischeri surface-bound DNA aptamer Respectively.
Detection method and growth inhibition method of Vibrio ficus-free live bacteria using an aptamer binding to the surface of Vibrio ficus-indica live cells
The present invention relates to a composition for detecting Vibrio fischeri live cells containing the selected DNA aptamer as an active ingredient.
As demonstrated in the following specific embodiment of the present invention, the DNA aptamer of the present invention specifically binds to the surface of a viroid of a viroid, so that it is useful for detecting the presence of viable viroid in a sample.
The present invention relates to a kit for detecting Vibrio fischeri live cells comprising the selected DNA aptamer as an active ingredient.
The kit may be in the form of a chip in which the DNA aptamer is immobilized on a chip, or the DNA aptamer may be in the form of a microarray immobilized on a substrate. The DNA aptamer can be immobilized on a chip or a substrate using a method known in the art.
According to a specific embodiment of the present invention, a chip or a substrate is modified by introducing streptavidin, biotinylated at the 5 'end of the DNA aptamer, Or binding with streptavidin introduced on a substrate.
As used herein, the term " microarray " means an array (array) in which DNA nucleic acid material is attached at a high density to a specific region of a substrate. As used herein, the term " substrate of a microarray " refers to a support having a suitable rigid or semi-rigid and may be, for example, a glass, membrane, slide, filter, chip, wafer, fiber, magnetic bead or non- , Gels, tubing, plates, polymers, microparticles, and capillaries. The DNA aptamers of the present invention are arranged and immobilized on the substrate. Such immobilization is carried out by a chemical bonding method or a covalent bonding method such as UV. For example, DNA oligonucleotides can be attached to glass surfaces modified to include epoxy compounds or aldehyde groups, and can also be bound by UV on polylysine coating surfaces. In addition, the DNA oligonucleotides may be bound to the substrate via linkers (e.g., ethylene glycol oligomers and diamines). The DNA aptamers of the present invention can be biotinylated, for example, and can be successfully coupled onto a substrate coated with streptavidin. The DNA aptamer of the present invention immobilized on a substrate can bind to and capture a viableifishi live cell, and the thus captured viableifishi live cell again uses a DNA aptamer that specifically binds to the viroid cell viable cell Capture can be visualized.
The kit of the present invention may further include a label or labeling material for use in detecting Vibrio ficifoliate in a sample.
The present invention includes a step of contacting a sample expected to contain Vibrio fischeri live cells with the DNA aptamer described above and identifying a Vibrio fischeri live cell bound to the DNA aptamer To a method for detecting Vibrio fischeri live cells.
Since the DNA aptamer of the present invention specifically binds to the surface of the Vibrio ficus, the DNA aptamer can be used to detect the Vibrio ficus live bacteria by contacting a sample expected to contain the Vibrio ficus live bacteria . The detection of the Vibrio fisuri bound to the DNA aptamer can be performed based on a method of detecting a DNA aptamer and a Vibrio ficus-binding complex. In order to facilitate detection of the complex, DNA aptamers may be used as a fluorescent substance, such as fulorescein, Cy3 or Cy5; Radioactive materials, or chemicals, such as nucleotides labeled with biotin or modified with primary amines.
(I) a DNA aptamer as described above; And (ii) a DNA aptamer-antimicrobial substance complex comprising an antimicrobial substance conjugated to the DNA aptamer through a linker.
In addition, the present invention relates to a method for inhibiting the growth of Vibrio fischeri by contacting the DNA aptamer-antimicrobial substance complex with a sample expected to contain Vibrio fischeri live cells.
When a DNA aptamer-antimicrobial substance complex is prepared by conjugating an antimicrobial substance to the DNA aptamer of the present invention and the complex is contacted with a sample expected to contain Vibrio fischeri live cells, It is possible to effectively inhibit the growth of Vibrio fischeri by increasing the number of contact between the microbes.
In the present invention, the conjugation between the DNA aptamer and the antimicrobial substance can be carried out through a suitable method known in the art, and preferably, the conjugation can be performed through a linker. The linker may be, for example, SPDP (N-succinimidyl 3- (2-pyridyldithio) propionate), S-Hynic (succinimidyl-6-hydrazino-nicotinamide), or S-4FB (N-succinimidyl- If the antimicrobial substance to be bound is a protein, the N-terminus of the protein and the amin group at the 3'-terminal or 5'-terminal of the DNA aptamer and the biotin group may be used. Antibacterials and DNA aptamers may be conjugated, but are not limited thereto.
The antimicrobial agent that can be used in the present invention may be a substance that exhibits an antimicrobial activity against Vibrio ficus, for example, an inorganic antibacterial agent derived from an inorganic compound, an organic antibacterial agent derived from an organic compound, a nano-silver, antimicrobial substances derived from animals such as nano-catalyst, bioceramics, metal salts having ion emitters effect, natural antimicrobial substances, propolis, lactoferrin, lysozyme and chitosan, and microorganisms such as nisin and polylysine But are not limited to, antimicrobial agents derived therefrom.
The advantages and effects of the present invention are summarized as follows:
(I) The present invention allows DNA aptamers to bind to the surface of Vibrio fischeri live cells at a high specificity.
(Ii) Using the DNA aptamer of the present invention, it is possible to detect and confirm the presence of Vibrio ficus-producing bacteria in the sample.
(Iii) Use of a DNA aptamer-antimicrobial substance complex in which an antimicrobial substance is conjugated to the DNA aptamer of the present invention can inhibit the growth of Vibrio ficus-indica and effectively inhibit the biofilm formed by the bacterium have.
FIG. 1 shows the results obtained by amplifying random DNA aptamer using PCR method, then selectively recovering only ssDNA using streptavidin agarose resin, and amplifying only by PCR. Lane 1: 100 bp DNA size marker; Lane 2: amplification of DNA aptamers by PCR; Lane 3: After amplification by PCR method, only ssDNA was recovered using streptavidin agarose resin.
Figure 2 quantitatively measures the eluent concentration of the Vibrio Fischeri surface-bound DNA aptamer group recovered in each round of the 10 rounds of the SELEX procedure for the preparation of the Vibrio Fischeri surface-bound DNA aptamer using a nano-drop This is a result.
Figure 3 shows the results of the first (panel (a), second (panel (a), and panel (b) panels for each of the Vibrio Fischeri surface- bound DNA aptamer candidates obtained in the selected rounds after the SELEX process for producing the Vibrio Fischeri surface- b)) is the result of quantitatively measuring the eluent with nano-drop.
4 shows the expected secondary structure of the Vibrio Fischeri surface-bound DNA aptamer VFCA-03 using an m-fold program.
FIG. 5 is a view showing a process of fixing Vibrio Fischeri surface-bound DNA aptamer to the surface of a sensor chip SA coated with streptavidin and binding Vibrio ficus-producing bacteria.
FIG. 6 is a graph showing the results obtained by fixing the Vibrio Fischeri surface-bound DNA aptamer on the surface of a sensor chip SA (GE healthcare, USA) coated with streptavidin, Genes, Escherichia coli, and Vibrio parahaemolyticus were screened to confirm the specificity of Vibrio Fischeri surface-bound DNA aptamer.
FIG. 7 is a schematic diagram of a rapid kit for visual confirmation of the presence of vibrio on a sample using Vibrio Fischeri surface-bound DNA aptamer. After attaching DNA aptamers to gold nanoparticles (gold nanoparticles) and flowing the sample into the sample pad, the Vibrio Fischeri and DNA aptamer in the sample are combined and the other lines of the test line and the Vibrio Fischeri in the test line are fixed. . This confirms the presence of Vibrio fischeri on the sample.
FIG. 8 shows that when the Vt. Bacteriophage surface-bound DNA aptamer is bound to lactoferrin, which is an antimicrobial substance, when lactoferrin-DNA aptamer conjugated with lactoferrin and DNA aptamer is added to the bacterial culture solution, the contact chance between the target strain and lactoferrin is increased .
Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these embodiments are only intended to further illustrate the present invention and that the scope of the present invention is not limited by these embodiments.
Example
Example 1: Preparation of a DNA aptamer pool
1-1. Synthesis of primer and DNA aptamer pool
In order to produce Vibrio Fischeri surface-bound DNA aptamer that specifically binds to Vibrio fisheri, the site of 5'-ATACCAGCTTATTCAATT and AGATAGTAAGTGCAATCT-3 ` ATACCAGCTTATTCAATT-N40-AGATAGTAAGTGCAATCT-3 '(SEQ ID NO: 14) having 40 consecutive random nucleotides at the center and a ratio of dA: dG: dC: dT = 1.5: 1.15: 1.25: (Forward primer: 5'-ATACCAGCTTATTCAATT-3 '(SEQ ID NO: 5) was prepared by ordering a forward primer capable of amplifying the DNA library and a biotinylated reverse primer to recover single strand DNA in Bioneer, Korea 15), biotinylated reverse primer: 5'-biotin-AGATTGCACTTACTATCT-3 '(SEQ ID NO: 16).
1-2. Amplification of DNA aptamer pool using PCR technique
The synthesized primer and any DNA library having 40 consecutive random sequences were amplified using PCR (Bioneer, Korea). The PCR reaction composition for the amplification of the 76 bp DNA library was composed of 5 μl of 10 × PCR buffer, 4 μl of each 2.5 mM dNTP mixture, 2 μl of 10 μM forward primer, 2 μl of biotinylated reverse primer, 1 - 2 μl of the library, 0.3 μl (1 unit / μl) of Ex Taq polymerase (TaKaRa, Japan) and 34.7 - 35.7 μl of distilled water. The PCR reaction conditions were first denatured at 94 ° C for 5 minutes, followed by 20 cycles of reaction at 94 ° C for 30 seconds, 52 ° C for 30 seconds, and 72 ° C for 30 seconds, followed by extension at 72 ° C for 5 minutes Reaction was used. After the PCR reaction, 3 μl was taken, and the band was observed at 76 bp using 2% agarose gel. The identified DNA was recovered from the DNA aptamer pool using a PCR purification kit (Qiagen, USA).
1-3. Production and purification of DNA aptamer pool ssDNA
As described above, 200 μl of a biotinylated dsDNA aptamer pool at the 5 'end was boiled at 85 ° C. for 5 minutes to prepare a biotinylated dsDNA aptamer pool as a ssDNA aptamer pool at the 5' end, and then rapidly cooled . Streptavidin agarose resin was added to 100 μl of the dsDNA aptamer pool and reacted with streptavidin at room temperature for 1 hour or more. Subsequently, centrifugation (4 ° C, 13,000 rpm) was performed for 10 minutes to precipitate streptavidin agarose resin, thereby removing one of the biotinylated dsDNA aptamer pools through streptavidin-biotin binding, A tamer pool was made. Then 200 μl of the upper layer was transferred to a new tube to ensure a pure ssDNA aptamer pool. And generally utilized the PCI treatment and ethanol precipitation method used in the industry. In this method, 200 μl of the same amount of PCI was added to the transferred ssDNA aptamer pool, and the lipids and proteins remaining in the ssDNA were removed by stirring. After centrifugation (4 ° C., 13,000 rpm) was performed for 15 minutes, To remove phenol. After that, 600 μl of triplicate of the amount of DNA was added to ethanol, 20 μl of 1/10 times 3M sodium acetate and 1 - 2 μl of tRNA were added, and then 1 μl of a 1 ° C deep freezer Hour. The DNA was then precipitated by centrifugation (4 ° C, 13,000 rpm) for 20 minutes, and the upper layer was removed. After drying in a heat block at 85 ° C, 100 μl of distilled water was added to obtain a ssDNA aptamer pool.
1-4. PAGE for identifying ssDNA aptamer pool
The pool of ssDNA aptamer recovered via streptavidin and biotin binding was confirmed by electrophoresis on 0.5X TBE acrylamide gel. As the composition of the 0.5X TBE (Tris-borate-EDTA) acrylamide gel, 3.75 ml of 40% acrylamide and 0.75 ml of 10X TBE buffer were added, and 15 ml of distilled water was added. Then, 135 μl of 10% APS (Ammonium per sulfate) TEMED (Tetramethylethylenediamine). The acrylamide gel was mixed with 10 μl of ssDNA aptamer pool and 2 μl of loading dye (DNA loading), and a 100 bp DNA marker was used as a ladder marker. The acrylamide gel for identification of ssDNA aptamer pool for polyacrylamide gel electrophoresis (PAGE) was electrophoresed on a power supply (Major Science, USA) at 150 V (volt) for 45 minutes. The electrophoretic acrylamide gel was observed by irradiating ultraviolet light with Gel-doc (Bio-Rad, USA) after ETBR solution was stained for 5 minutes (see FIG. 1).
1-5. Three-dimensional structure formation of DNA aptamer
100 μl of the ssDNA aptamer pool prepared in advance to form a three-dimensional structure of the ssDNA aptamer pool as confirmed by PAGE in the above Example 1-4 was added to 100 μl of the same amount of 2 × LBS medium, Heated for 5 minutes in a heat block preheated to induce denaturation of the ssDNA aptamer pool. The denatured ssDNA was slowly cooled at room temperature for 2 hours or more to induce its own three - dimensional structure formation.
Example 2: Vibrio fischeri ( Vibrio fischeri ) Preparation of live-cell for the production of live-cell-bound DNA aptamer
2-1. Preparation and Culture of Vibrio Fischeri
To produce a Vibrio ficus-type live-coupled DNA aptamer, Vibrio ficusi was obtained through culture. As the culture medium, LBS (1% tryptone in 10 mM Tris-HCl, 0.5% yeast extract and 2% NaCl) was used and cultured in a 37 ° C agitation incubator for 12 hours. In addition, to remove other substances that are formed during cultivation to bind only to the live cells, the cultured Vibrio fischeri was washed with a washing solution having a composition of 10 mmol / L of Tris base, 0.85% of NaCl and pH 8.0. In the washing method, the cultured Vibrio fischeri was first centrifuged (4 ° C, 13,000 rpm) for 10 minutes to precipitate Vibrio fischeri and remove the supernatant. Thereafter, 200 μl of washing solution was added, and the surface of the Vibrio fischeri was removed by pipetting and centrifugation (4 ° C, 13,000 rpm) was performed for 10 minutes to precipitate the Vibrio fischeri and remove the upper layer. The above procedure was repeated three times to clean the Vibrio facial surface.
2-2. Negative selection and cultivation of other bacteria for comparison of binding force
Negative selection using Escherichia coli and Bacillus subtilis was performed to remove non-specific aptamer candidates binding to other strains of the Vibrio ficus-specific binding DNA-aptamer candidates specifically binding to Vibrio ficusi . In order to compare the binding force of DNA aptamer to Vibrio ficusi in SPR measurement, binding force was measured using other cultured strains.
2-2-1. E. coli ( Escherichia coli ) Culture and securing
Negative selection LB broth was used as a culture medium for Escherichia coli, which is a target strain. 5 ml of LB broth was inoculated and cultured at 37 占 폚 for 12 hours. Then, the cultured Escherichia coli was centrifuged (4 캜, 13,000 rpm) for 10 minutes to precipitate and the upper layer was removed. After that, 200 μl of washing solution was added, and the surface of E. coli was washed by pipetting, followed by centrifugation (4 ° C., 13,000 rpm) for 10 minutes to precipitate and remove the upper layer. The above procedure was repeated three times to wash the surface of E. coli.
2-2-2. Bacillus subtilis ( Bacillus subtilis ) Culture and securing
Negative selection LB broth was used as a culture medium for Bacillus subtilis, a target strain. 5 ml of LB broth was inoculated with Bacillus subtilis and cultured at 37 DEG C for 12 hours. Subsequently, the cultured Bacillus subtilis was centrifuged (4 캜, 13,000 rpm) for 10 minutes to precipitate and the upper layer was removed. After that, 200 μl of washing solution was added, and the surface of Bacillus subtilis was washed by pipetting and centrifugation (4 ° C., 13,000 rpm) was performed for 10 minutes to precipitate and remove the upper layer. The above procedure was repeated three times to clean the Bacillus subtilis surface.
2-2-3. Listeria monocytogenes ( Listeria monocytogenes ) Culture and securing
NA broth was used as a culture medium for Listeria monocytogenes, a strain for measuring the non-specificity of aptamer in SPR measurement. 5 ml of NA broth was inoculated with Listeria monocytogenes and cultured at 37 占 폚 for 12 hours. The cultured Listeria monocytogenes was then centrifuged (4 ° C, 13,000 rpm) for 10 minutes to precipitate and the upper layer was removed. Thereafter, the surface of Listeria monocytogenes was washed with pipetting by adding 200 세척 of wash solution, centrifuged (4 캜, 13,000 rpm) for 10 minutes to precipitate and the upper layer was removed. The above procedure was repeated three times to wash the Listeria monocytogenes surface.
2-2-4. Vibrio parahaemolyticus ( Vibrio parahaemolyticus ) Culture and securing
NA broth containing 3% NaCl was used as a culture medium for Vibrio parahaemolyticus, which is a strain for confirming non-specificity of aptamer in SPR measurement. 5 ml of NA broth containing 3% NaCl was inoculated with Vibrio parahaemolyticus and cultured at 37 DEG C for 14 hours. The cultured Vibrio parahaemolyticus was then centrifuged (4 ° C, 13,000 rpm) for 10 minutes to precipitate and the upper layer was removed. After that, 200 μl of the washing solution was added, and the surface of the Vibrio parahaemolyticus was removed by pipetting, followed by centrifugation (4 ° C., 13,000 rpm) for 10 minutes to precipitate and remove the upper layer. The above procedure was repeated three times to clean the surface of Vibrio parahaemolyticus.
Example 3: Fabrication of Vibrio Fischeri surface binding aptamer that specifically binds to Vibrio fischeri
3-1. Screening and securing candidates for DNA aptamers that specifically bind to Vibrio ficus.
After washing the Vibrio ficus strains obtained by the methods described in Examples 1 and 2, 200 μl of the ssDNA aptamer pool having a three-dimensional structure of its own was put in a Thermo mixer (Eppendorf, USA) at 4 ° C. and 500 rpm And reacted for 1 hour. Then, the tube was taken out and centrifuged (4 ° C, 13,000 rpm) for 10 minutes to precipitate the Vibrio fischeri having the ssDNA aptamer pool and remove the supernatant. Vibrio ficus-associated DNA aptamers were precipitated by the weight of Vibrio fischeri, and unbound ssDNA aptamers remained in the upper layer.
To remove the ssDNA aptamer pool that was not bound with the Vibrio fischeri among the precipitated Vibrio fischeri and Vibrio ficus-associated viable DNA aptamer candidates, 200 μl of the washing solution was added and pipetted, followed by centrifugation (4 ° C, 13,000 rpm ) For 10 minutes to precipitate Vibrio fischeri and remove the supernatant. The above procedure was repeated three times to remove the ssDNA aptamer pool that uniquely binds to Vibrio fischeri. Then, 100 μl of a DNA aptamer elution solution composed of 10 mM Tris (pH 7.5) and 1 mM EDTA was added to the precipitate. Thereafter, the cells were allowed to react for 5 minutes in a heating block preheated to 85 ° C, followed by centrifugation (4 ° C, 13,000 rpm) for 10 minutes to precipitate the Vibrio fischeri, and the Vibrio fischeri specific binding DNA aptamer The supernatant was transferred to a new tube to recover a candidate DNA aptamer that specifically binds to Vibrio ficusi. The above procedure was repeated twice to recover 100 μl of each DNA aptamer pool.
3-2. Negative selection of candidate Vibrio ficus-specie-specific DNA binding aptamers
Negative screening was performed to eliminate nonspecifically binding DNA aptamers after the sixth round of selection. Escherichia coli and Bacillus subtilis live cells were obtained by the methods described in Examples 1 and 2. First, 200 μl of the ssDNA aptamer pool which formed the three-dimensional structure of Escherichia coli was added, and the mixture was added to a Thermo mixer (Eppendorf, USA) And reacted at 500 rpm for 1 hour. The tubes were then taken out and centrifuged (4 ° C, 13,000 rpm) for 10 minutes to precipitate the non-specific ssDNA aptamer pool-bound E. coli and to obtain supernatants. The obtained supernatant was added to the prepared Bacillus subtilis and reacted for 1 hour at 4 ° C and 500 rpm in a Thermo mixer (Eppendorf, USA). The aptamer bound to E. coli and Bacillus subtilis, but not the target strain, was removed during the recovery of the supernatant.
3-3. Purification and Elution Concentration of Vibrio Fischeri Species-Specific Binding DNA Aptamer Candidate
To remove proteins and lipids remaining in the vibrio ficus-specific enzyme-linked DNA aptamer pool and recover only DNA, the PCI treatment and the ethanol precipitation method conventionally used in the industry were used. 100 μl of the PCI solution was treated and mixed with 100 μl of each recovered DNA aptamer pool. After centrifugation (4 ° C, 13,000 rpm) was performed for 15 minutes, 100 μl of the supernatant was transferred to a new tube. After that, 300 μl of ethanol (100% ethanol) was added, and 10 μl of 3M sodium acetate and 1 μl of tRNA were added and reacted at -70 ° C for 1 hour or more in a deep freezer. Then, DNA was precipitated by centrifugation (4 ° C, 13,000 rpm) for 20 minutes, and the upper layer was removed. After drying in a heat block at 85 ° C, 50 μl of distilled water was added, Respectively. 1 μl of the obtained DNA aptamer pool was measured in a nano-drop to confirm the DNA concentration.
Example 4: Selection of DNA aptamer candidates showing optimal binding force with Vibrio fischeri
4-1. Screening and securing each round DNA aptamer pool
50 μl of ssDNA aptamer pool of each round obtained after preparing ssDNA using each elongated DNA aptamer pool of each round was prepared in the same manner as described above The mixture was placed in a Vibrio Fischeri sediment and reacted with a Thermo mixer (Eppendorf, USA) at 4 ° C and 500 rpm for 1 hour. Then, the tube was taken out and centrifuged (4 ° C, 13,000 rpm) for 10 minutes to precipitate the Vibrio fischeri having the structure-forming ssDNA aptamer pool attached thereto. After removing the upper layer, 200 μl of the washing solution was pipetted and centrifuged (4 ° C, 13,000 rpm) for 10 minutes. The upper layer was removed and washed three times. 100 μl of the elution solution was added and heated at 85 ° C for 5 minutes, followed by centrifugation (4 ° C, 13,000 rpm) for 10 minutes To obtain eluted DNA aptamers for each round.
4-2. Analysis of each round elution DNA aptamer pool using nano-drop
In order to remove proteins and lipids remaining in each round of DNA aptamer pools and to recover only DNA, they were purified using the PCI treatment and the ethanol precipitation method conventionally used in the art. After 1 ㎕ of DNA aptamer pool of each round was measured by nano-drop and DNA concentration was checked and negative selection was completed, 8 rounds of the highest concentration of aptamer candidates (See FIG. 2).
Example 5: Vibrio Fischeri surface-bound DNA aptamer candidate analysis that specifically binds to Vibrio ficusi in selected rounds
5-1. T-vector cloning and selection for DNA aptamer candidates
For the selection of DNA aptamer candidates for the selected 8 rounds, the reaction composition for the amplification of the 8 round DNA aptamer pool was 5 μl of 10X PCR buffer, 4 μl of each 2.5 mM dNTP mixture, 10 μl of
5-2. Grouping of candidate Vibrio ficus-specific DNA-binding DNA aptamer candidates through Clustal-X
The DNA aptamer sequence inserted through cloning among the sequences for each colony was identified and sequenced and analyzed using the Clustal-X program. Groups were formed for each sequence, and the sequence similarity between the sequences was analyzed, and 13 sequences grouped into 6 groups including 7 identical sequences were obtained (see Table 1 below).
(SEQ ID NO: 1)
(SEQ ID NO: 2)
(SEQ ID NO: 3)
(SEQ ID NO: 4)
(SEQ ID NO: 5)
(SEQ ID NO: 6)
(SEQ ID NO: 7)
(SEQ ID NO: 8)
(SEQ ID NO: 9)
(SEQ ID NO: 10)
(SEQ ID NO: 11)
(SEQ ID NO: 12)
(SEQ ID NO: 13)
Example 6: Evaluation of the affinity of the candidate group of Vibrio ficus-indicie live cell specific binding DNA aptamer
6-1. Purification of Vibrio ficus-indica candidate strains
In order to purify the previously obtained Vibrio ficus-specific binding DNA aptamer candidate group, a pure Vibrio ficus-specific binding DNA aptamer candidate group purified by the PCI treatment and the ethanol precipitation method was obtained.
6-2. Evaluation of affinity of Vibrio Fischeri specific binding DNA aptamer candidates using nano-drop
In order to evaluate the affinity for the purified Vibrio Fischeri specific binding DNA aptamer candidates, 1 μl of each Vibrio Fischeri specific binding DNA aptamer candidate group obtained was measured by nano-drop and the DNA concentration was determined 1 2 and 3 candidates of DNA aptamer candidates, which were the highest concentrations in the case of tea extract and second elution, were selected (see FIG. 3).
Example 7: Prospective structural analysis of selected DNA aptamers
7-1. Analysis of expected secondary structure of each DNA aptamer candidate group
In order to confirm the expected structure of VFCA-03, which has the highest specificity among selected Vibrio Fischeri specific binding DNA aptamers, the expected structure was analyzed using the M-fold program of http://mfold.rna.albany.edu/ . As a structural analysis condition, the sequence of DNA aptamer was designated as a linear strand and the temperature of the structure was set at 25 ° C, which is room temperature. The expected structure was prepared by designating the concentration of sodium capable of affecting the structure to 0.3M, the concentration of LBS as the culture medium (see FIG. 4).
Example 8: Preparation of DNA aptamer and Vibrio fischeri for SPR measurement
8-1. Preparation of DNA aptamer for SPR (Surface Plasmon Resonance) measurement
PCR was carried out using the method described in Example 1 except that 10 pM biotinylated reverse primer and 10 pM biotinylated forward primer and 10 pM reverse primer were added in place of 10 pM biotinylated reverse primer in the PCR sample . The following procedure was followed in accordance with Example 1 to perform PCR purification. As PCR was performed, asymmetric PCR was used to construct ssDNA. 10 μl of 10X PCR buffer, 8 μl of each 2.5 mM dNTP mixture, 10 μl of 10 pM biotinylated forward primer, 1 μl of 10 μM reverse primer, 10 μl of template DNA library, 10 μl of Ex Taq polymerase (1 unit / 쨉 l) and 60.5 쨉 l of distilled water (TaKaRa, Japan). The PCR reaction conditions were first denaturation at 94 ° C for 5 minutes, followed by 15 cycles of reaction at 94 ° C for 30 seconds, 52 ° C for 30 seconds, and 72 ° C for 30 seconds, followed by extension at 72 ° C for 5 minutes Was used. After that, pure DNA aptamer was purified and secured using PCI treatment and ethanol precipitation method conventionally used in the art.
8-2. Construction of ssDNA aptamer
After 1 μl of the obtained ssDNA was added to the nano-drop and the ssDNA concentration was measured, 99 μl of 2X LBS was added to the remaining 99 μl of the ssDNA aptamer, and the mixture was heated at 85 ° C for 5 minutes, Lt; / RTI > Thereafter, it was diluted in 1X LBS to prepare a DNA concentration of 25 μM.
8-3. Preparation of Vibri-Fischeri for Surface Plasmon Resonance (SPR) Measurements
Vibrio fischeri were cultured on the basis of the method described in Example 2 for SPR measurement. Thereafter, the surface of the live cells was washed with the washing solution according to Example 2.
Example 9: Selection of optimal DNA aptamer by SPR measurement
9-1. Fixed DNA aptamer on sensor chip SA
Streptavidin immobilized with dextran was activated on the gold valve by the method described in the protocol provided by GE healthcare. To fix the DNA aptamer on the surface of the activated sensor chip SA, the flow rate of the sample was set at 10 μl / min. One of the four channels of the sensor chip SA did not attach an aptamer for the test, and combined the selected aptamers with only two, three, and four channels. As a binding condition of DNA aptamer, a series of steps of 1 minute inoculation at a flow rate of 10 μl / min was repeated three times each. After that, HBS-EP buffer was flown at a flow rate of 10 / / min for 10 minutes to stabilize the bound ssDNA aptamer.
9-2. Combination of Vibrio Fischeri
1 ml of the cultured medium containing the prepared Vibrio fischeri was transferred to a tube, and then a series of procedures of flowing the sensor chip SA at a flow rate of 5 μl / min for 20 minutes were repeated twice. During this process, the binding force was confirmed through comparison and analysis of Sensogram of
Example 10: Evaluation of specificity of optimal Vibrio ficus-lyase-binding DNA aptamer
10-1. Affinity measurement of Vibrio parahaemolyticus
Vibrio parahaemolyticus was cultured for comparison of binding strength with Vibrio fischeri. Cultivation of Vibrio parahaemolyticus was carried out according to the method described in Example 2 above. After that, 1 ml of the culture was transferred to a tube, and then a series of procedures of flowing the sensor chip SA at a flow rate of 5 μl / min for 20 minutes were repeated twice. Thereafter, a series of procedures of flowing 50 mM NaOH at a flow rate of 10 쨉 l / min for 5 minutes were repeated twice to remove the combined vibrio parahaemolyticus and ssDNA aptamer, followed by
10-2. Affinity measurement of Listeria monocytogenes
Listeria monocytogenes was cultured for comparison of binding strength with Vibrio fischeri. Cultivation of Listeria monocytogenes was carried out according to the method described in Example 2 above. After that, 1 ml of the culture was transferred to a tube, and then a series of procedures of flowing the sensor chip SA at a flow rate of 5 μl / min for 20 minutes were repeated twice. Thereafter, a series of procedures of flowing 50 mM NaOH at a flow rate of 10 쨉 l / min for 5 minutes were repeated twice to remove the bound Listeria monocytogenes and ssDNA aptamer, followed by ssDNA aptamer three times Lt; / RTI >
10-3. Affinity measurement of Shgellasonei
For comparison of binding strength with Vibrio fischeri, Shigella sonoei were cultured. The cultivation method of Shigella sognae was carried out according to the method described in Example 2 above. After that, 1 ml of the culture was transferred to a tube, and then a series of procedures of flowing the sensor chip SA at a flow rate of 5 μl / min for 20 minutes were repeated twice. Thereafter, a series of procedures of flowing 50 mM NaOH at a flow rate of 10 쨉 l / min for 5 minutes were repeated twice to remove the bound sh sgellasone and ssDNA aptamer, and then the ssDNA aptamer was washed three times Lt; / RTI >
10-4. Measurement of affinity of E. coli
Escherichia coli was cultured for comparison of binding strength with Vibrio fischeri. Culture of E. coli was performed according to the method described in Example 2 above. After that, 1 ml of the culture was transferred to a tube, and then a series of procedures of flowing the sensor chip SA at a flow rate of 5 μl / min for 20 minutes were repeated twice. Thereafter, a series of procedures of flowing 50 mM NaOH at a flow rate of 10 쨉 l / min for 5 minutes were repeated twice to remove the bound Escherichia coli and the ssDNA aptamer, and the ssDNA aptamer was bound 3 times as described above Table 3).
Example 11: Development of Rapid kit platform for rapid detection of Vibrio fischeri
In order to industrially utilize the Vibrio Fischeri specific binding DNA aptamer manufactured and secured through the above examples, in order to confirm the detection method using the Vibrio Fischeri specific binding DNA aptamer and its applicability, We have developed a rapid kit platform to check the detection capability of the Rapid Kit. The rapid kit can utilize streptavidin-biotin binding, and a brief schematic diagram of the construction of the rapid kit is shown in FIG. In order to detect Vibrio fischeri using a Vibrio FISHERY Rapid Kit, a Vibrio Fischeri specific binding DNA aptamer and streptavidin were immobilized on a nitrocellulose membrane (see Fig. 7 (a)). Subsequently, in order to confirm that the sample containing Vibrio ficus-containing bacteria was actually processed and detected, the amino group was substituted at the 5'-end of the Vibrio ficus-specific binding DNA aptamer, and 10 nm carboxylated gold nanoparticles (Carboxylated gold (Amine coupling kit) buffer solution for immobilization of amine-bound Vibrio ficus-specific DNA-binding aptamer to nanoparticles, and treated at 25 ° C for 1 hour. Then, the prepared amine-bonded Vibrio ficus- It can be confirmed by inducing reaction with the app tamer. The vibrio ficus-specific binding DNA aptamers immobilized on the nanoparticles prepared in this procedure were mixed with the samples containing Vibrio fischeri. The mixture was treated at 25 ° C for 1 hour and then loaded in 10X loading buffer (2.5% Triton X-100, 500 mM Tris-HCl, 10
In the presence of Vibrio fischeri in the sample, two lines appear (refer to FIG. 7 (b)). In the absence of Vibrio fischeri, only one line is displayed. In order to confirm the ability of the prepared Vibrio fischeri detection kit, If not, we can prepare a sample that does not have Vibrio fischeri and a sample that contains Vibrio fischeri to check the color of control line and test line. It was designed to confirm that the Vibrio Fischeri detection kit produced through the above experiment was made normally.
Example 12: Optimal Vibrio FISHERY BACTERIAL BINDING A DNA aptamer was conjugated to lactoferrin, an antimicrobial substance, to control Vibrio Fischeri control and inhibit biofilm formation
12-1. Vibrio ficrier live-binding DNA Aptamer and lactoferrin conjugated with
A DNA aptamer with an amine group was synthesized to bind lactoferrin with a DNA aptamer that specifically binds to Vibrio fischeri. The synthesized DNA aptamer was conjugated after activating both ends using Biolinker SPDP reagent (N-succinimidyl 3- (2-pyridyldithio) propionate, Thermo scientific). The SPDP junction was performed according to the method described in the protocol provided by the manufacturer (Thermo scientific). The concentration of lactoferrin was fixed at 10 ㎎ / ㎖ and the concentration could be adjusted according to the target strain.
12-2. Modified aptamer treatment of Vibrio ficus live cells
Vibrio ficus-free bacteria were cultured according to the method described in Example 2. [ The cultured viable cells were again inoculated into three 100 ml LBS medium, and compared with the negative control (no negative control), lactoferrin, and lactoferrin conjugated with aptamer, Respectively. The cultures were continuously cultured for 24 hours in a 30 ° C agitating incubator, and the ODs (Optical Density) were measured every 1 hour by using the Bradford assay commonly used in the art, and the degree of inhibition of growth was determined by the growth curve have.
12-3. Possible inhibition of Vibrio fischeri growth and inhibition of biofilm formation
Through the experiments performed in Example 12-2, it was confirmed that the growth curve of Vibrio ficuli was inhibited by deriving a growth curve using OD measured over time. In this experiment, the addition of lactoferrin conjugated with aptamer inhibited the growth of Vibrio fischeri, followed by lactoferrin and negative control. A method for confirming the formation amount of the biofilm can be confirmed by performing a staining method using crystal violet. Vibrio fischeri was inoculated into 5 ml of LBS medium and incubated overnight at 30 ° C. After diluting to an OD 600 value of 0.1, 200 μl of each was added to a 96-well plate. After sufficient incubation for 24 hours, the supernatant was removed and the wells were washed 2-3 times with 1x PBS. Thereafter, the cells were stained with 200 μl of 1.0% crystal violet for 20 minutes to be stained with a biofilm and a cell extract. The stained wells were thoroughly washed 2-3 times with 1x PBS and then dried to remove cell extracts except for the biofilm attached to the well surface. 200 μl of 100% ethanol was used to decolorize the crystal violet dyed on the biofilm and the sample was measured at OD 550 to quantify the dyed biomass. In this experiment, it was confirmed that lactoferrin conjugated with aptamer has the best biofilm inhibiting ability similar to growth inhibition.
Example 13: Study for industrial application of biofilm formation inhibition ability of finally selected Vibrio Fischeri surface-bound DNA aptamer
In order to industrially utilize the ability of the Vibrio Fischeri surface-bound DNA aptamer to inhibit the biofilm formation of the Vibrio Fischeri surface biofilm in an environment infected with Vibrio fischeri, The binding DNA aptamers inhibit the growth of Vibrio ficusi and control the biofilm. It is expected that biofilm can be limited in diverse environments by utilizing the power of various samples.
While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is to be understood that the same is by way of illustration and example only and is not to be construed as limiting the scope of the present invention. Accordingly, the actual scope of the present invention will be defined by the appended claims and their equivalents.
<110> Chungbuk National University Industry Academic Cooperation Foundation <120> DNA Aptamer Specifically Binding to Surface of Living Cell of Vibrio fischeri and Uses Thereof <130> MP14-0207 <160> 16 <170> Kopatentin 2.0 <210> 1 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> DNA aptamer <400> 1 ggtcgtgtgg acttgcgatt tcggtttggt gtggttggtg 40 <210> 2 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> DNA aptamer <400> 2 gggcatgtgg acttgcgatt tcggtttggt gtggttgggg 40 <210> 3 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> DNA aptamer <400> 3 gggcatgtgg gcttgcgatt tcggtttggt gtggttgggg 40 <210> 4 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> DNA aptamer <400> 4 tggcatgtgg acttgcgatt tcggtttggt gtggttgggg 40 <210> 5 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> DNA aptamer <400> 5 gggcatgtgg acttgcggtt tcggtttggt gtggttgggg 40 <210> 6 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> DNA aptamer <400> 6 gggcgtgtgg acttgcgatt tcggtttggt gtggttgggg 40 <210> 7 <211> 39 <212> DNA <213> Artificial Sequence <220> <223> DNA aptamer <400> 7 gggcatgtgg acttgcgatt tcggttggtg tggttgggg 39 <210> 8 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> DNA aptamer <400> 8 gggcatgtgg acttgcgact tcggtttggt gtggttgggg 40 <210> 9 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> DNA aptamer <400> 9 agacagcata gcactgtaac gattggtttg gtgtggttgg 40 <210> 10 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> DNA aptamer <400> 10 ccagaattgg tgggtcgact gctggtgtcc tataaagggg 40 <210> 11 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> DNA aptamer <400> 11 cgtgggcggt agagccaatg ctactggagc gcgtatccta 40 <210> 12 <211> 41 <212> DNA <213> Artificial Sequence <220> <223> DNA aptamer <400> 12 gtcggagagc ccggccgtct gctcgtaagt actatcatag c 41 <210> 13 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> DNA aptamer <400> 13 gcggacggat agtaaatagc ccatgtacgc tgctggcgtc 40 <210> 14 <211> 76 <212> DNA <213> Artificial Sequence <220> <223> DNA library for preparing DNA aptamer <400> 14 ataccagctt attcaattnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnag 60 atagtaagtg caatct 76 <210> 15 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> PCR primer <400> 15 ataccagctt attcaatt 18 <210> 16 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> PCR primer <400> 16 agattgcact tactatct 18
Claims (11)
(a) contacting a sample suspected of containing Vibrio fischeri live cells with the DNA aptamer of any one of claims 1 to 4; And
(b) Identifying the Vibrio fischeri live cells bound to the DNA aptamer.
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| PCT/KR2015/011328 WO2016072653A1 (en) | 2014-11-06 | 2015-10-26 | Dna aptamer specifically binding to surface of live cells of vibrio fischeri, and use thereof |
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