KR20110093024A - Dna aptamer specifically binding to anthrax toxins and uses thereof - Google Patents

Abstract

PURPOSE: A DNA aptamer which specifically binds to anthrax lethal toxin is provided to ensure immune response without side effects. CONSTITUTION: A DNA aptamer(ssDNA aptamer) specifically binds to anthrax lethal toxin prtein selected from a group consisting of nucleic acid having a base sequence of sequences 1-4. A DNA microarray for diagnosing anthrax contains at least one DNA aptmer selected from the group consisting of nucleic acid having a base of sequence numbers 1-4.

Description

DNA Aptamer specifically binding to anthrax toxin protein and its use {DNA APTAMER SPECIFICALLY BINDING TO ANTHRAX TOXINS AND USES THEREOF}

The present invention relates to a DNA aptamer specifically binding to anthrax toxin protein, anthrax treatment agent and biosensor using the same.

An aptamer refers to an oligomer, which is a specific molecular recognition material that can be artificially synthesized. Aptamers consist of a single nucleotide sequence, DNA or RNA, and have a specific affinity and specificity for a variety of target substances due to the specific three-dimensional structure that the nucleotide sequence can produce.

By applying the technique of finding the most suitable DNA molecule from a highly diverse DNA library (> 10 ¹⁵ or more DNA having various sequences), DNA specific to the target material can be obtained. In general, a DNA library is a mixture of DNA molecules having more than 30 random sequences and defined sequences at both ends. This method is called SELEX (Systemic Evolution of Ligands by Exponential Enrichment). SELEX technology randomized oligonucleotides synthesized with a small number of in-objects in a target material having the affinity complex from the nucleotide Trojan (in screening in vitro ).

Since the introduction of SELEX, an aptamer discovery technology, many researchers have developed aptamers for a variety of targets, including proteins, peptides, nucleic acids, vitamins, antibiotics, metal ions and cells. SELEX technology is also developing and accelerating.

The aptamers discovered by SELEX technology can be used as biomaterial sensing materials such as antigen-antibody reactions. The aptamer's structure can change when the aptamer binds to the target material, and the structural flexibility of the aptamer can be applied to various fields. In particular, it is used as a method for improving the sensitivity of the sensor when developing the biosensor. Aptamers can also be used as microarrays or microchips, as well as to immobilize the target material in a stable state.

As mentioned earlier, aptamers are single-stranded nucleic acids that bind with high affinity and specificity to specific protein targets. Therefore, there is little immune reaction (immunogenesity) that is a disadvantage of the antibody, there is an advantage that can be easily chemically synthesized within a short period. In addition, aptamers are attracting attention as the next-generation pharmaceuticals to replace the antibody market because various modifications can be easily made.

Anthrax is a Gram-positive bacillus Bacillus anthracis ) is a common infectious disease caused mainly by livestock but also by humans. This anthrax has long been developed as a biological weapon. In recent years, they have been deployed in practice, and in 1990, they even received anthrax vaccine against US troops stationed overseas during the Gulf War in 1990 and 1998. In this case, anthrax is also a major concern for the general public.

Anthrax caused by anthrax infections is known to have a very high mortality rate. Anthrax treatment, like other bacterial infections, has three approaches.

The first is vaccination to block anthrax infections in advance. The second is to neutralize infected anthrax with antibiotics. The final method is to detoxify the major disease toxins produced by infected anthrax.

Once anthrax is infected, it is ideal to administer antibiotics and antidote to neutralize germs and toxins at the same time. Currently, however, the development of antidote compared to antibiotics is insignificant, and it is mainly dependent on antibiotics.

Korea also responds by classifying anthrax as a legal epidemic. In fact, there have been reports of deaths of people who have eaten livestock and lung meat. Therefore, the need for control of anthrax has emerged.

Antibiotics that kill anthrax have already been developed, but the drug has the drawback that it has no effect on the anthrax toxin already produced. And anthrax vaccines are also difficult to use because of side effects. Moreover, it is very important to detect and carry out mass treatment in the early stages of the development of anthrax with high mortality.

In addition, considering that more than half of domestic meat consumption is imported through the United States, which has a high bioterrorism risk (anthrax), a thorough quarantine system for livestock imports is urgently needed. However, in the case of livestock products in the early and advanced stages of the disease symptoms do not appear, there is a problem that the concentration of the disease-causing bacteria is very low to diagnose the disease and difficult to quarantine a large amount of livestock products.

In general, it is known that the pathogenicity of anthrax is determined by toxin production ability and capsular formation. Proteins that affect the toxin producing ability of anthrax are protective antigen (PA), edema factor (EF) and lethal factor (LF). Each of them is not toxic, but defense antigen (PA) and edema factor (EF) combine to form edema toxin (EdTx, edema toxin), and defense antigen (PA) and lethal factor (LF) bind to lethal toxin (LeTx, ethal toxin).

PA binds to receptor proteins (Anthrax toxin receptor (ATR)) on the surface of infected cell lines, such as macrophages, and is then cut into PA20 and PA63 by furin family protease, which binds to LF or EF as heptamers heptamerize. It is known to play the role of a toxin delivery channel to transport them into cells.

These complexes enter into cells through the endocytosis mechanism, and when the pH of the endosomes decreases, structural changes occur in the PA63 heptamer and pores on the endosomal membrane. As a result, LF or EF is released into the cytoplasm and has a toxic effect. . Therefore, PA is of interest for the prevention and treatment of anthrax. Therefore, many studies on the method of inhibiting anthrax toxin by using anthrax toxin protein PA and the like.

However, no detection agent with high sensitivity, rapid and in situ adaptability to anthrax toxin protein, or a detection kit with characteristics such as high physicochemical stability, sensitivity, specificity and applicability has not been suggested.

Accordingly, the present inventors have developed a DNA aptamer as a new material that can be used for the diagnosis and treatment of anthrax.

It is an object of the present invention to provide a DNA aptamer that specifically binds anthrax toxin protein.

Another object of the present invention is to provide an anthrax therapeutic agent using the DNA aptamer.

Another object of the present invention is to provide a DNA microarray and biosensor for diagnosing anthrax using the DNA aptamer.

The present invention provides a DNA aptamer specifically binding to anthrax toxin protein, which is selected from the group consisting of nucleic acids having the nucleotide sequences of SEQ ID NOs: 1-4.

The anthrax toxin protein may be protective antigen (PA) 63 or lethal factor (LF).

DNA aptamer according to the present invention is a single-stranded DNA represented by SEQ ID NO: 1 to 4, specifically binds to anthrax toxin protein PA or LF. The DNA aptamer binds to PA or LF, which is a target substance, to form a three-dimensional structure, wherein the function of the target protein, PA or LF, is inhibited. Specifically, PA may be inhibited from binding edema factor (EF) to form edema toxin (EdTx, edema toxin), or PA may bind with LF to form lethal toxin (LeTx, ethal toxin).

As described above, when the DNA aptamer of the present invention binds to the anthrax toxin protein, the PA63 is heptamerized, thereby preventing binding to LF or EF. As a result, PA63 may not function as a toxin delivery channel, which may inhibit the effects of anthrax toxin.

Aptamers are short single-stranded oligonucleotides that can form three-dimensional structures that can bind to target materials with high affinity and specificity. In most cases, such aptamers not only specifically bind to the target protein but also effectively inhibit the function, and thus can be used to determine the function of the target protein.

In order to achieve the above object, the present invention was to select a single-stranded DNA aptamer having a binding capacity corresponding to the antigen-antibody reaction as a diagnostic target of the toxin transport protein showing anthrax toxicity. Specifically, SELEX screening was performed to select oligonucleotides that bind to anthrax toxin proteins such as PA63, and then analyzed their binding power to select DNA aptamers.

The SELEX method of the present invention is a method known in the art. Briefly, it is a method of determining the DNA binding sequence of a molecule by selecting and amplifying a DNA or RNA having a high binding capacity to a specific molecule in a randomly synthesized collection of DNA or RNA.

Using the SELEX method, four kinds of low-molecular aptamers that specifically bind to anthrax toxin protein with specific affinity were identified, and are represented by SEQ ID NOs: 1 to 4. DNA aptamers of SEQ ID NOS: 1-4 show high binding affinity for PA63 or LF.

In the present invention, a low molecular weight nucleic acid aptamer is used as a target material to overcome the disadvantages of the antibody. These small molecule nucleic acid aptamers are small in size and can easily form three-dimensional structures that allow them to bind to the target material. In addition, the aptamer according to the present invention is relatively simple in mass production and weak in toxicity.

The present invention also provides an anthrax therapeutic agent containing as an active ingredient at least one DNA aptamer selected from the group consisting of nucleic acids having a nucleotide sequence of SEQ ID NOs: 1 to 4, which inhibits the interaction between anthrax toxin proteins.

Inhibition of interaction between the anthrax toxin protein may be to inhibit the mutual binding between PA63 and LF or PA63 and EF. The mechanism of mutual binding between PA63 and LF or EF has been described above.

The aptamer according to the present invention can be used in combination with proteins as well as organic molecules, inorganic molecules, carbohydrates, cells. Therefore, the aptamer according to the present invention can be used for targeted therapy by acting as an antagonist to anthrax toxin protein. Specifically, the aptamer of the present invention is used as an active ingredient of anthrax therapeutics.

The anthrax therapeutic agent may include a therapeutically effective amount of aptamer or salt thereof, and a pharmaceutically acceptable carrier or diluent.

Anthrax therapeutics, including the aptamers of the present invention, have great affinity and specificity for their targets when the aptamer therapeutics degrade in the body of a patient or subject while reducing the deleterious side effects caused by naturally occurring non-nucleotide nucleotide substituents.

The anthrax therapeutic agent containing the aptamer of the present invention as an active ingredient may further contain one or more active ingredients exhibiting the same or similar functions.

Anthrax therapeutics of the present invention generally contain an effective amount of therapeutically active ingredient dissolved or dispersed in a pharmaceutically acceptable medium. Pharmaceutically acceptable media or carriers include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like. The use of such media and medicaments in pharmaceutically active substances is well known in the art. Supplementary active ingredients can also be incorporated into the therapeutic compositions of the invention.

Pharmaceutically acceptable carriers may be used in combination with saline, sterile water, Ringer's solution, buffered saline, dextrose solution, maltodextrin solution, glycerol, ethanol and one or more of these components, as necessary. Other conventional additives such as buffers and bacteriostatic agents can be added. In addition, diluents, dispersants, surfactants, binders and lubricants may be additionally added to formulate injectable formulations such as aqueous solutions, suspensions, emulsions and the like. ,

The preparation of the therapeutic agents according to the invention can be carried out using methods known to those skilled in the art. Typically, the therapeutic composition is a liquid solution or suspension; As an injectable drug in solid form suitable for solution or suspension in liquid before injection; As a tablet or other solid for oral administration; As a time release capsule; Or any form of therapeutic agent used today, including eye drops, creams, lotions, salves, inhalants and the like.

Therapeutic agents comprising the aptamers of the invention are preferably parenteral (eg, applied to the intravenous, subcutaneous, intraperitoneal, topical or visual route). Dosage varies depending on the weight, age, sex, health condition, diet, time of administration, method of administration, rate of excretion and severity of disease of the patient. Aptamers of the invention are useful for treatment at dosage levels ranging from about 0.001 μg to 100 mg per kg body weight. The dosage can be administered in a single dose or can be divided into several doses. In general, the desired dosage should be administered at set intervals over a long period of time, at least over several weeks, although longer dosing periods may be required.

In another aspect, the present invention provides an anthrax diagnostic DNA microarray comprising at least one DNA aptamer selected from the group consisting of nucleic acids having a nucleotide sequence of SEQ ID NO: 1 to 4.

The "DNA microarray" refers to a microarray in which DNA groups are immobilized at a high density on a substrate, and the DNA groups are immobilized in a predetermined region, respectively. Methods of making such microarrays are well known in the art.

By "diagnostic" is meant identifying the presence or characteristic of a pathological condition. For the purposes of the present invention, the diagnosis is to identify the presence or characteristics of anthrax.

DNA aptamer according to the present invention is excellent in specificity and high binding capacity to the target can be utilized to diagnose the presence or absence of anthrax invented in the target sample. The diagnostic DNA microarray of the present invention may further include a buffer or reaction solution that stably maintains the structure or physiological activity of the DNA aptamer. In addition, to maintain stability, it may be provided in a powder state or dissolved in a suitable buffer.

In addition, in order to facilitate identification, detection and quantification of the DNA aptamer of the present invention bound to anthrax toxin protein, the DNA of the present invention is labeled with a protein, an organic molecule, an inorganic molecule, or a carbohydrate, as described above. Can be provided.

In addition, the DNA aptamer of the present invention may be provided in a form coated on the surface of the plate. In this case, anthrax can be diagnosed by inoculating target cells directly on the plate and reacting the cells under appropriate conditions, and then observing the binding of anthrax toxin protein and DNA on the surface of the plate.

Experimental procedures, reagents and reaction conditions that can be used in the above methods can utilize those conventionally known in the art, which is obvious to those skilled in the art.

In addition, the present invention provides an anthrax diagnostic biosensor comprising at least one DNA aptamer selected from the group consisting of nucleic acids having the nucleotide sequences of SEQ ID NOs: 1-4.

The biosensor may further include a fluorescent material.

The fluorescent material may be used without any limitation what is used in the art. Specific examples include, post-drawn (OliGreen ^®), but are the fluorescein (fluorescein), tetramethyl rhodamine (tetramethylrhodamine), Cy5, Cy3 and Texas red (Texas Red) or the like, but are not necessarily limited thereto.

The aptamer according to the present invention may be used as a biosensor, specifically, may be used as an “electrochemical based biosensor” or “fluorescence based biosensor”.

Electrochemical-based biosensors are systems that measure the change in current or resistance that occurs when anthrax toxin protein binds to an aptamer after fixing the aptamer to an electrode.

Specifically, guanine has been found to have an electron transfer mechanism among bases present on DNA. However, even if guanine is present in the aptamer DNA, it is generally electrochemically silent at the applied voltage, so it is necessary to perform amplified electrochemical detection using an external mediator, an inorganic metal compound. Catalytic oxidation of guanine can be detected. Ru (bpy) ₃ ²⁺ , [Fe (CN) ₆ ] ^4, etc. are used as mediators that play such a role. Thus, an aptamer of the present invention, which selectively binds only to anthrax toxin protein and does not require a labeling process, can easily diagnose anthrax infection.

Fluorescence-based biosensors refer to a system that quantitatively detects the binding of an aptamer and a target substance by using a fluorescent substance. The fluorescent material is labeled on the aptamer to be used as a biosensor, or the aptamer and the target material are combined to be used as a biosensor. Specifically, a biosensor may be manufactured using a fluorescent material, for example, a fluorescent dye called OliGreen ^® (Molecular probe Inc.). The oligrin is a pigment used for quantification of single-stranded DNA, and may bind to double-stranded DNA to fluoresce. That is, if there is no DNA in the aqueous solution, oligrins cannot bind to the DNA and thus do not exhibit fluorescence. By using this property, the aptamer and anthrax toxin protein according to the present invention can be combined with the fluorescent material, and then the intensity of fluorescence can be measured to detect the anthrax toxin protein. Since the oliglin can adhere to a large number of DNA of one molecule, it can be confirmed whether or not amplification compared to the fluorescence intensity when there is no anthrax toxin protein.

Since the DNA aptamer of the present invention specifically binds to anthrax toxin protein, the DNA aptamer may be used as an anthrax therapeutic agent, anthrax diagnostic DNA microarray, and a biosensor. When the DNA aptamer of the present invention is used for the treatment and diagnosis of anthrax, there is almost no side effect such as an immune response that occurs in the existing antigen-antibody reaction.

Figure 1 shows the results of the isolation and purification of PA63 protein according to an embodiment of the present invention.
2 is a TEM image showing the results after induction of heptamerization of PA proteins. (a) is the PA63 protein and the arrow shows the heptamer ring structure, and (b) the PA83 protein used as a control did not show the heptamer ring structure.
Figure 3 is a schematic diagram of the Cy3-aptamer produced for analysis of the binding force with the aptamer binding to anthrax toxin protein.
4A to 4D are graphs showing the binding force between the aptamer of SEQ ID NOs: 1 to 4 and an anthrax toxin protein PA63.
5 is a graph showing the binding force between the aptamers and anthrax toxin proteins PA63 and LF of SEQ ID NOS: 1 to 4 according to the present invention.
Figure 6 shows the three-dimensional structure of the aptamer of SEQ ID NOS: 1 to 4 in accordance with the present invention.
Figure 7 schematically shows a biosensor using a fluorescent material produced according to an embodiment of the present invention.
Figure 8 shows the result of detecting the combination of anthrax toxin protein and aptamer using the fluorescent biosensor (black line is 10 nM DNA aptamer (SEQ ID NO: 1) + 15 nMPA (protective antigen which is anthrax toxin protein) ) + OG (OliGreen) solution, blue line shows 15 nM PA + OG solution, and red line shows 10 nM DNA aptamer + PA + OG, with 40 nM PA added).
9 is a graph showing the change in fluorescence when PA is added by concentration using the fluorescence biosensor (the black line is without PA, the blue line is 10 nM PA, and the red line is 25 nM PA). , And green lines indicate when 40 nM PA was added).
10 is a graph showing whether the BSA (control) protein and aptamer bind using the fluorescent biosensor (black line is a solution containing 10 nM aptamer + OG, red line is 10 nM aptamer + OG + Solution with 40 nM BSA, blue line when 40 nM BSA (Bovine Serum Albumin) + OG).

Hereinafter, preferred examples are provided to help understanding of the present invention, but the following examples are merely to illustrate the present invention, and various changes and modifications can be made within the scope and spirit of the present invention. It is apparent to those skilled in the art that such modifications and variations are within the scope of the appended claims.

Example  One: PA63  Mass production and separation of proteins

In order for PA to function as an toxin-carrying protein that exhibits anthrax toxicity, it must be bound to the surface of the cell line to be infected and then converted into amino terminal sites (PA20) and carboxy terminal terminals (PA63) by proteolytic enzymes. Can be. That is, after conversion to the form of PA63, PA63 must form a heptamer form so that LF or EF can be competitively bound. The combined polymers enter the cell and become an endosome. Only the PA63 moiety, to which these toxin polymers bind, was cloned. To this end, an anthrax strain ( Bacillus anthracis sterne ) was distributed from the National Institute of Disease Control, and the genomic DNA of the strain was isolated and the PA63 gene was amplified by PCR. PCR amplification was performed as follows: upstream primers were amplified using 5'-AAGAGCTCGGGATCCGTGCGGGCGGTCATGGT-3 'and downstream primers using 5'-AAGGTCGACTTATGAGTTAATAATGAACTTAATCTG-3'. To facilitate cloning, a base sequence of 5 '-(BamHI): reverse 3' (NotI), a restriction enzyme recognition site, was attached to each of the primer ends. Using a DNA amplifier, amplification was performed 25 times in a cycle of a total denatured 95 ° C, 2 minutes, denatured 95 ° C, 45 seconds, annealing 55 ° C, 1 minute, and elongation 68 ° C for 3 minutes. It was then inserted into the pET22b (Novagen) vector cut with BamHI and NotI restriction enzyme and cloned according to the manufacturer's manual (pET22-PA).

This cloned PA63 expression plasmid was transformed into E. coli BL21 (DE3), which is an expression host, to induce PA63 production for 24 hours under conditions of 0.5 mM IPTG and 18 ° C. The transformation method is briefly described as follows: First, 5 μl of DNA was added to 100 μl of competitive cells, mixed well, and left on ice for 30 minutes. Then heat shock was applied at 42 ° C. for 90 seconds. In order to prevent contamination, the alcohol lamp was first turned on, and then 800 µl of LB medium was added and incubated at 37 ° C for 45 minutes. Finally it was applied to the LBA plate and incubated overnight in a 37 ° C. incubator.

The bacterial cells were harvested by centrifugation and examined for solubility of the proteins. As a result, most of the proteins were insoluble. Therefore, for the isolation and purification of PA63 protein, only intracellular insoluble protein was obtained and then dissolved in denaturing conditions (6M guanidine-HCl, 20 mM Tris, pH8.0, 300 mM NaCl, 5 mM imidazole and 1 mM BME). The Ni-Sepharose column was then passed through to bind the PA63 protein. Then, while rebounding the denatured PA, PA63 is refolded and 500 mM imidazole, slowly denatured through linear gradient (reduced concentration of urea) while bound to the column. Eluted under a linear gradient.

The obtained protein was dialyzed in a solution of PBS (137 mM NaCl, 2.7 mM KCl, 10 mM Na ₂ HPO ₄ , 2mMKH ₂ PO ₄ , pH 7.4) to remove excess imidazole. Purity of the obtained protein was confirmed through 12% SDS / PAGE, the amount was confirmed using the Bradford (Bradford) reaction solution was obtained 3 mg / L protein (Fig. 1).

Then, transmission electron microscopy (TEM) and native gels (gel electrophoresis under undenatured conditions) were used to observe the refolding of the PA63 protein and the heptamer formation of PA63. Was run. PA63 is known to be heptamerized to bind to LF, EF to play the role of a toxin delivery channel. Therefore, by checking whether the PA63 protein separated and purified by the above method was heptamerized, it was confirmed whether the structure of the protein was normally refolded after denaturation purification. When PA63 was treated according to the environmental conditions that heptamerization can occur (0.5 ug of purified PA63 and 20 mM Hepes (pH 7.4) and reacted at room temperature for 1 hour), PA63 formed a heptamer ring structure. It confirmed (FIG. 2 (a)).

Example  2: Anthrax  Specifically binding to toxin proteins Single strand DNA Aptamers  making

Single-stranded DNA aptamer capable of specifically binding to anthrax toxin protein PA63 from a random single-stranded DNA library (GenoTech Co., Deajeon, Korea) consisting of approximately 7.2 × 10 ¹⁴ DNA molecules was selected by SELEX method It was.

In order to discover specific binding aptamers using the PA63 protein purified in Example 1, first, a random DNA library is required. To this end, a single stranded DNA library (5'-GCGCGGATCCCGCGC (N ₃₀ ) CGCGCGAAGCTTGCG-3 ') containing a randomly constructed base sequence ((N) ₃₀ ) was used in a total of 60 bases (Integrated DNA Technologies). Primers were prepared and synthesized to amplify the template DNA library. The DNAs were amplified by PCR with the forward primer "5'-Biotin-GCGCGGATCCCGCGC-3 '(including BamHI site)" and the reverse primer "5'-CGCAAGCTTCGCGCG-3' (including HindIII site)". To obtain only single-stranded DNA from the amplified DNA, streptavidin (New England Biolabs) was used to bind streptavidin-biotin ssDNA, followed by 6M urea gel to isolate only ssDNA and precipitated in ethanol. Single-stranded DNA was identified.

In vitro selection of oligonucleotides for screening aptamers that specifically bind to PA63 using single-stranded DNA ( in vitro SELEX, a selection of oligonucleotides, was performed. The concentration filter was used as a method modified from the existing SELEX method (see Table 1 below). When proteins and aptamers bind, they are larger than the size of the membrane, so they do not fall down and remain on top, so that unbound single-stranded DNA can be separated and separated.

To this end, PA63 and single-stranded DNA were reacted at room temperature for about 30 minutes using ultrafiltration (100 kDa cutting-membrane-containing micron, Millipore), and the single-stranded DNA did not bind to PA63 through centrifugation. It was separated by falling down, and was unable to pass through the membrane, which was on the membrane, which was amplified by PCR, and then hung on a 2.5% agarose gel to obtain only PA63-DNA, and proceeded to the next round. At this time, ssDNA was added to the bound PA63 to obtain ssDNA expected to bind with high binding strength to PA 63 through protein and gene concentration, binding time, and binding conditions (Table 1).

Four DNA aptamers were finally selected through a total of eight screening and amplification procedures for PA63. The base sequences of these DNA aptamers are shown in Table 2 below.

SEQ ID NO: Sequence (5 '→ 3') One CAGACCGTAAGGGATGCCGCCT AAACACC 2 GATGTGGGTGTAGTTGGAGGGTAAACGTT 3 GGAATAGAGACAAAGCCACTCATATGAGT 4 CGCACTGAAGTGGGATACCGCCTAAACGG

Example  3: Anthrax  Through cross-reaction between toxin proteins Aptamer  Binding force analysis

In order to determine the binding force to the anthrax toxin protein of the base sequence found in Example 2, the binding force analysis was performed using PA63. For binding analysis, binding affinity of four aptamers bound to PA63 was analyzed using a fluorescence microplate reader (Molecular Devices).

More specifically, the ssDNA of SEQ ID NOS: 1 to 4 was amplified using a primer synthesized using a fluorophore called fluorophore (Cy 3) to identify Cy3-ssDNA (FIG. 3). After determining the DNA concentration obtained, put the aptamer of SEQ ID NOS: 1 to 4 according to the conditions of each step in the PA63-coupled plate (96 well black plate, SPL), and measure the fluorescence intensity of Cy3 connected to the bound aptamer By confirming the binding force of the aptamer. The fluorescence signal intensity was measured and displayed according to the concentration, and the apparent Kd value was determined according to the saturation curve (FIG. 4).

Kd values were obtained using known methods (Kim JM et.al , (2006) Journal of Microbiology and Biotechnology 16,1784-1790.) Specifically, PA63 protein (0.5 g / well) was added to 20 mM Hepes ( pH7.4) Heptamerization was induced by reacting the solution for 30 minutes at room temperature. Then, the protein was fixed in SPL (96 well-black Immunoplate) at room temperature for 1 hour, washed three times with 1 × TBST buffer, and 2% BSA was added for 1 hour for blocking. . After washing the SPL containing the PA protein 5 times and then amplified by diluting the amplified ssDNA (Cy3 labeled) aptamer of SEQ ID NOS: 1 to 4, and the PA protein and ssDNA at room temperature for 1 hour Combined. The conjugate was then washed three times with 20 mM Hepes (pH7.4), and then the saturation curves were determined by measuring the Cy3-labeled ssDNAs at 545 nm excitation and 572 nm fluorescence emission. And Kd value was determined.

As shown in FIG. 4, the aptamer according to the present invention shows the binding force at the nanomolar level. In particular, the two eptamers showed an excellent binding force of 1 nM or less, thereby confirming the binding force corresponding to that of the existing antibody-antigen reaction.

Example  4: Anthrax  Bound to toxin protein Aptamer  Binding force analysis

The binding affinity of the aptamer of SEQ ID NOS: 1-4 and LF, another anthrax toxin protein, was performed. Specifically, in order to determine whether the aptamers of SEQ ID NOs: 1 to 4 obtained through the screening bind to a protein in which anthrax protein occupies a large portion or aptamers to bind essential amino acids of all proteins, anthrax toxin protein Phosphorus PA and LF, and BSA (Bovine Serum Albumin) as a control to compare the binding force in the same manner as in Example 3.

To this end, proteins were fixed on 96-well black plates at the same concentration (1.0 g / well), and then washed, and then the binding force was analyzed by putting single-stranded DNA aptamers (100 nM) while only proteins were fixed to the plates.

In particular, as shown in Figure 5, put the PA63, LF and BSA protein on the surface of the plate and then put the aptamer and the aptamer binding to it was confirmed using a fluorescent. As a result, the aptamers of SEQ ID NOS: 1 and 2 relatively bound to PA63, and SEQ ID NOs: 3 to 4 were better bound to LF.

Example  5: DNA Aptamer  Confirmation of solid structure

As described above, four kinds of DNA aptamers having excellent binding strength in the range of nanomolar (10 ^-9 M) among aptamers bound to the anthrax toxin protein obtained through the present invention can be obtained. The three-dimensional structure of the aptamers was examined through the “M-fold program”. The results are shown in FIG. As shown in Figure 6, the DNA aptamer of SEQ ID NO: 1 to 4 can be seen that the structure is easily modified to easily bind to anthrax toxin protein.

Example  6: DNA Aptamer  Fluorescence analysis based biosensor

A biosensor using an OG (OliGreen ^® , Molecular Probe Inc.) fluorescent dye was prepared. The OG is a dye used for quantification of single-stranded DNA, and may bind to double-stranded DNA to fluoresce. If there is no DNA in the aqueous solution, it does not fluoresce. By using the characteristics of the OG it is possible to make a biosensor to quantitatively determine the extent to which the ssDNA aptamer according to the invention binds to the PA protein. OG can attach to a large number of molecules of one molecule, so it can be checked whether the fluorescence intensity is amplified.

15 nM PA protein was added to the ssDNA aptamer aqueous solution (10 nM ssDNA aptamer) of SEQ ID NO: 1 and reacted with 0.05 ng / ml OG (the reagent dissolved in 1 ml of DMSO to 0.05 ng / ml). 10 mM Tris-HCl, 1 mM EDTA, pH 7.5) was added), and the change in fluorescence was measured using a fluorescence microplate reader (Molecular Devices). As a control, OG was added to PA 15 nM.

As shown in FIG. 8, the solution in which OG was added to 10 nM aptamer + 15 nM PA shows large fluorescence at 525 nm (10 nM PAA1, black spectrum). When OG was added to PA (15 nM), fluorescence was not shown (15 nM PA, blue spectrum).

In addition, to test the fluorescence of the PA protein and the aptamer itself in the absence of the fluorescent substance OG, when the fluorescence was measured after adding 40 nM PA to 10 nM PAA1, the fluorescence of 525 nm was almost quenched. It can be seen that quenching, that is, fluorescence disappearance (PAA1 + 40 nM PA, red spectrum). This shows that the fluorescence of the protein and the aptamer itself is not emitted.

Next, as shown in FIG. 9, when 10, 10, 25, and 40 nM of PA were added to 10 nM aptamer (A1) and 0.05 ng / ml of OG was added, the fluorescence was measured. It can be seen that the intensity of is decreased.

The reason why the intensity of the fluorescence decreases is that, as the amount of PA added increases, the relatively large PA protein first binds to the aptamer, so that the probability of OG binding to the aptamer decreases, and the fluorescence signal is caused by protein interference. Is lowered. In addition, as the concentration of the protein increases, the probability of binding of the aptamer and the protein increases, so that the portion of the OG can bind to the aptamer, and thus the fluorescence intensity decreases as the concentration of the protein is increased. That is, it can be seen that the binding force of the DNA aptamer and anthrax toxin protein according to the present invention is high. Through this DNA aptamers of the present invention can be used as a probe of the fluorescence-based biosensor.

As a control experiment, a solution obtained by adding 10 nM of DNA aptamer of SEQ ID NO: 1 to 10 nM PA protein (PAA1); A solution in which 40 nM BSA protein was further added to a solution containing 10 nM PA protein and 10 nM DNA aptamer of SEQ ID NO: 1 (PAA1 + 40 nm BSA); And fluorescence intensity was measured by adding 0.05 ng / ml of OG, respectively, to a solution consisting only of BSA protein (BSA only). As a result, as shown in Figure 10, the fluorescence was hardly reduced even under the conditions of PAA1 + 40nm BSA. That is, the BAS protein does not fluoresce by OG because the aptamer according to the present invention does not bind to the BSA protein. Therefore, it can be seen that the biosensor using the aptamer according to the present invention can specifically detect only anthrax toxin protein.

The DNA aptamer of the present invention can be actively used in various fields such as aptamer therapeutics, medical diagnostic techniques, biosensors, drug delivery systems, molecular imaging material analysis and isolation techniques.

<110> Industry-University Cooperation Foundation Hanyang University <120> DNA APTAMER SPECIFICALLY BINDING TO ANTHRAX TOXINS AND USES          THEREOF <130> P09-7191 <160> 4 <170> KopatentIn 1.71 <210> 1 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> DNA aptamer to anthrax toxins <400> 1 cagaccgtaa gggatgccgc ctaaacacc 29 <210> 2 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> DNA aptamer to anthrax toxins <400> 2 gatgtgggtg tagttggagg gtaaacgtt 29 <210> 3 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> DNA aptamer to anthrax toxins <400> 3 ggaatagaga caaagccact catatgagt 29 <210> 4 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> DNA aptamer to anthrax toxins <400> 4 cgcactgaag tgggataccg cctaaacgg 29

Claims (8)

DNA aptamer specifically binding to anthrax toxin protein selected from the group consisting of nucleic acids having the nucleotide sequences of SEQ ID NOs: 1-4.
The DNA aptamer specifically binding to anthrax toxin protein according to claim 1, wherein the anthrax toxin protein is PA63 or LF.
An anthrax therapeutic agent containing as an active ingredient at least one DNA aptamer selected from the group consisting of nucleic acids having the nucleotide sequences of SEQ ID NOs: 1 to 4, which inhibits the interaction between anthrax toxin proteins.
The agent of claim 3, wherein the inhibition of interaction between the anthrax toxin protein inhibits the mutual binding between PA63 and LF or PA63 and EF.
Anthrax diagnostic DNA microarray comprising at least one DNA aptamer selected from the group consisting of nucleic acids having the nucleotide sequences of SEQ ID NOs: 1-4.
Anthracnose diagnostic biosensor comprising at least one DNA aptamer selected from the group consisting of nucleic acids having the nucleotide sequences of SEQ ID NOs: 1-4.
The biosensor for diagnosing anthrax according to claim 6, wherein the biosensor further comprises a fluorescent material.
The method of claim 7, wherein the fluorescent material is post-drawn (OliGreen ^®), being selected from the group consisting of fluorescein (fluorescein), tetramethyl rhodamine (tetramethylrhodamine), Cy5, Cy3 and Texas red (Texas Red) Biosensor for diagnosing anthrax.

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