KR100995426B1 - DNA aptamer specifically binding to arsenic and uses thereof - Google Patents

Abstract

The present invention utilizes DNA aptamers to solve environmental pollution problems caused by harmful heavy metals, and relates to selection of arsenic binding DNA aptamers that specifically bind to arsenic, among other heavy metals. Specifically, a process for preparing a arsenic binding DNA aptamer and a arsenic binding DNA aptamer screening process, and various industrial / environmental applications of the arsenic binding DNA aptamer obtained therefrom and a composition comprising the arsenic binding DNA aptamer It is about.

Arsenic binding, DNA aptamers, heavy metals, microarrays, kits, SELEX

Description

DNA aptamer specifically binding to arsenic and uses according to arsenic

The present invention relates to the activity of DNA aptamers and their aptamers specifically binding to arsenic, which is a toxic substance, and more specifically, to our daily lives such as colorants, pesticides (pesticides, herbicides, etc.), waste batteries, and preservatives. In order to utilize DNA aptamers in arsenic removal and arsenic detection chips, which are included in various forms, arsenic binding column preparation, arsenic binding DNA aptamer selection, and optimal arsenic binding DNA aptamer selection for arsenic binding DNA aptamer selection Process, optimal arsenic binding DNA aptamer characteristics and arsenic specificity, DNA aptamer column manufacturing process for removing arsenic contained in various sample solutions, various applications in the environmental field using the same and the arsenic binding DNA 결합 aptamer It relates to a composition.

At present, the global environment is rapidly being polluted due to the development of various industries, which causes the damage not only to humans but also to all animals and plants. In particular, humans are end consumers of the food pyramid and are unprotected from increasing carcinogenesis and birth defects due to the consumption of various toxic pollutants. In particular, arsenic is a highly toxic carcinogenic substance that greatly contaminates the surrounding environment, and is widely used for ingesting food based on soil or water containing arsenic contaminants, inhaling arsenic mixtures, and exposing skin. Influenced by the pathway, it shows fatal toxicity across various organs such as skin, respiratory system, digestive system and nervous system. On the other hand, arsenic exists in the natural environment such as soil and groundwater in the form of arsenic ion (+3, arsenite) or oxyanion of arsenate (+5, arsenate). It is known to be more thermodynamically stable. On the other hand, it is known to show a significant difference in solubility, mobility and toxicity according to the species of arsenic. In particular, toxicity depends on the oxidation state and the degree of classification of organic and inorganic forms, and reduction, inorganic forms are generally known to be more toxic than oxidation and organic forms. It is known that arsenic ions are 20 to 60 times higher in toxicity and mobility than arsenic ions.

As described above, various heavy metals, including arsenic with lethal toxicity, have emerged as serious environmental pollutants. Therefore, there is a need for an environment-friendly technology capable of effectively treating them.

As a technique for treating arsenic and various heavy metals included in the conventional environment, general solidification / stabilization, landfilling, soil cover, vitrification, plant purification, soil washing, and the like are known.

These methods collect contaminated soil or contaminated water and treat them with special solutions to purify them physically and chemically.In the case of a small contaminated area, they can effectively remove contaminants but cannot remove only specific contaminants. It has a disadvantage. In particular, contamination in the natural state is not only extensive, but effective arsenic removal methods have not been developed that are applicable to fluid field conditions such as various structure types, various pH, temperature, etc. at the pollution site.

Due to the high potential for application in the environment and industry, DNA aptamers have recently made a lot of efforts to produce and screen DNA aptamers that specifically bind to specific target materials, mainly by researchers from many developed countries. However, in the case of DNA aptamer produced / developed up to now, patents and core technologies are mostly monopolized by large research institutes and multinational corporations in developed countries, so research and application in domestic researchers and industries are limited. Therefore, it is very urgent to secure economically applicable aptamers and develop application technologies by securing a specific aptamer production and screening technology specifically binding to the target substance.

Korean Patent Publication No. 194-7000545 discloses aptamers specific for biomolecules and a method for their preparation, and RNA aptamers and their use are disclosed in PCT International Publication WO 2007/043784.

The present invention has been made in accordance with the above requirements, a method for producing arsenic-binding DNA aptamer specifically binding to arsenic, a heavy metal that is a major cause of serious environmental pollution in various environments of nature, arsenic binding DNA using the method Aptamer screening process, DNA aptamer column production method for removing arsenic in various sample solutions, various applications in the environmental field using the same, various industrial applications using the selected arsenic binding DNA aptamer and the It is intended to provide a composition comprising an arsenic binding DNA aptamer.

In order to solve the above problems, the present invention provides a DNA aptamer specifically binding to heavy metals.

The present invention also provides a composition for removing or detecting heavy metals containing the DNA aptamer as an active ingredient.

The present invention also provides a method for detecting a heavy metal in a sample using the DNA aptamer or a composition comprising the same.

The present invention also provides a microarray having a substrate on which the DNA aptamer is immobilized.

The present invention also provides a kit for heavy metal detection comprising the DNA aptamer.

The present invention also provides a arsenic binding sensor chip manufacturing method for arsenic binding DNA aptamer screening.

The present invention also provides a DNA aptamer screening method that specifically binds to heavy metals.

The present invention also provides a method for optimal arsenic binding DNA aptamer selection.

The present invention also provides a method for removing heavy metals in a sample using the DNA aptamer or a composition comprising the same.

The present invention utilizes DNA aptamers to solve environmental pollution problems caused by harmful heavy metals, and relates to selection of arsenic binding DNA aptamers that specifically bind to arsenic, among other heavy metals. Specifically, a process for preparing a arsenic binding DNA aptamer and a arsenic binding DNA aptamer screening process, and various industrial / environmental applications of the arsenic binding DNA aptamer obtained therefrom and a composition comprising the arsenic binding DNA aptamer Can be provided. Through the present invention, it was possible to propose a new type of method for removing heavy metals, which is the main culprit of environmental pollution, which secured the core technology for the technology of removing environmental pollutants, which is monopolized by developed countries and multinational corporations, thereby leading It is expected to be able to secure a wide range of industrial applications such as conversion to a next generation process that is competitive and environmentally friendly.

In order to achieve the object of the present invention, the present invention provides a DNA aptamer that specifically binds to heavy metals.

In the present specification, the aptamer refers to DNA having a high affinity for a target substance, and various target substances (proteins, sugars, stains, DNAs, metals) desired by a method called SELEX (Systematic Evolution of Ligands of Exponential enrichment). Aptamers for ions, cells, etc.).

The heavy metal may be arsenic (As), Cd, Pb, Cu, Fe, Mn, Ni, Co, Zn, Ca, Mg, etc., preferably As, Cd, Pb which is a heavy metal harmful to the human body, more preferably It is As.

DNA aptamer according to an embodiment of the present invention may have at least one nucleotide sequence of the base sequence of SEQ ID NO: 1 to SEQ ID NO: 8, preferably may have a nucleotide sequence of SEQ ID NO: 3. In addition, sequences complementary to the sequences can also be included within the scope of the present invention. In addition, variants of the sequences may be included within the scope of the present invention. A variant is a nucleotide sequence that changes in nucleotide sequence but has similar functional properties to the nucleotide sequences of SEQ ID NOs: 1-8. Specifically, the DNA aptamer gene has at least 70%, more preferably at least 80%, even more preferably at least 90%, most preferably at least 95% homology with the nucleotide sequences of SEQ ID NOs: 1-8 It may include a base sequence.

The "% sequence homology" for a polynucleotide is identified by comparing two optimally arranged sequences with a comparison region, wherein part of the polynucleotide sequence in the comparison region is the reference sequence (addition or deletion) for the optimal alignment of the two sequences. It may include additions or deletions (ie, gaps) as compared to not including).

DNA aptamers according to one embodiment of the invention may be single stranded DNA. Single-stranded DNA aptamers can form secondary structures that bind to heavy metals, preferably arsenic (see FIG. 7).

The present invention also provides a composition for heavy metal removal or detection comprising a DNA aptamer having at least one nucleotide sequence of SEQ ID NO: 1 to SEQ ID NO: 8 as an active ingredient. The heavy metal is preferably arsenic. Since the DNA aptamer of the present invention has a property of specifically binding to heavy metals, preferably arsenic, it may enable heavy metal removal or detection.

The present invention also provides a microarray having a substrate on which a DNA aptamer having at least one nucleotide sequence of SEQ ID NO: 1 to SEQ ID NO: 8 is immobilized. The microarray may be used for heavy metal detection, and the heavy metal may be preferably arsenic.

In the microarray of the present invention, "microarray" refers to a microarray in which oligonucleotide groups are immobilized at a high density on a substrate, and the oligonucleotide groups are immobilized in a predetermined region, respectively. Such microarrays are well known in the art. Microarrays are disclosed, for example, in US Pat. Nos. 5,445,934 and 5,744,305, the contents of which are incorporated herein by reference. The DNA aptamer is as described above.

The term "substrate" refers to any substrate to which a DNA aptamer can be attached under conditions that retain binding properties and keep the background level of binding low. Typically, the substrate may be a microtiter plate, membrane (eg, nylon or nitrocellulose) or microspheres (beads) or chips. Prior to application or fixation to the membrane, DNA aptamers can be modified to promote immobilization or improve binding efficiency. Such modifications may include homopolymer tailing, coupling with different reactive functional groups such as aliphatic groups, NH ₂ groups, SH groups and carboxyl groups, or coupling with biotin, hapten or protein.

The present invention also provides a kit for heavy metal detection comprising a DNA aptamer having at least one nucleotide sequence of SEQ ID NO: 1 to SEQ ID NO: 8. In the kit of the present invention, the heavy metal may be preferably arsenic. In addition, the kit of the present invention may further include a user manual describing the conditions for performing the optimal reaction.

The present invention also comprises contacting a sample suspected of containing a heavy metal with a DNA aptamer having at least one nucleotide sequence of SEQ ID NO: 1 to SEQ ID NO: 8 and detecting the binding of the heavy metal and DNA aptamer It provides a method for detecting a heavy metal in a sample comprising the step of. In the heavy metal detection method of the present invention, the heavy metal may be preferably arsenic, DNA aptamer may preferably have a nucleotide sequence of SEQ ID NO: 3.

Samples suspected of containing heavy metals may be, but are not limited to, colorants, pesticides (pesticides, herbicides, etc.), spent batteries, preservatives, river water, and the like. As a label of the aptamer for detecting the binding of the heavy metal and the DNA aptamer, a radioactive isotope, a fluorescent substance such as fluorescein, Cy3 or Cy5, or biotin may be used. It is available.

The present invention also comprises the steps of contacting a sample suspected of containing a heavy metal with a DNA aptamer having at least one nucleotide sequence of SEQ ID NO: 1 to SEQ ID NO: 8 to form a heavy metal and DNA aptamer complex and It provides a method for removing heavy metals in a sample comprising the step of removing the complex. In the heavy metal removal method of the present invention, the heavy metal may be preferably arsenic, DNA aptamer may preferably have a nucleotide sequence of SEQ ID NO: 3.

One embodiment of the method of the present invention comprises the steps of modifying one end with biotin to immobilize the DNA aptamer on the bead surface; Immobilizing the modified DNA aptamer on a bead surface; And inducing binding of the immobilized DNA aptamer and heavy metal. The beads may be coated with any one capable of binding to the biotin of the modified DNA aptamer, but may preferably be coated with streptavidin. When the surface coating of streptavidin is a biomolecule such as avidin that binds well to biotin is easily used, that is, using avidin-biotin binding commonly used in the art. In the heavy metal removal method of the present invention, preferably the bead is a magnetic bead (magnetic bead), may further comprise the step of removing the DNA aptamer bound to the heavy metal using a magnet. By binding DNA aptamers to magnetic beads to bind heavy metals, the bound DNA aptamer-heavy metal complexes can be separated using magnets, and the heavy metals can be selectively removed from the complexes. .

The present invention also provides

1) amplifying DNA aptamers using PCR and asymmetric PCR techniques and selecting ssDNA aptamers;

2) optionally fixing the heavy metal to the column; And

3) screening DNA aptamers that bind heavy metals through heavy metal binding columns and SELEX (Systematic Evolution of Ligands by Exponential Enrichment);

It provides a DNA aptamer screening method that specifically binds to a heavy metal comprising a.

In the DNA aptamer selection method of the present invention, asymmetric PCR is performed to amplify only single strand DNA (ssDNA) among dsDNAs amplified using PCR. Asymmetric PCR enables single stranded DNA to be obtained by performing PCR using only one of the forward and reverse primers, for example, the forward primer. The method for screening ssDNA aptamer, for example, amplifies dsDNA by attaching biotin to a reverse primer during PCR, and treating the amplification product with streptavidin to form a biotin-streptavidin complex to selectively remove the complex. As a result, only ssDNA aptamers can be selected. The obtained ssDNA aptamer may be treated with S1 nuclease to determine whether it is ssDNA (see FIG. 1).

DNA aptamer screening method that specifically binds to arsenic of the present invention is the step of coupling 4-aminophenylarsine oxide (4PAO) to affi-Gel 10 resin (Bio-rad, USA) and arsenic specific binding DNA using SELEX method Aptamer screening may include a method (see FIG. 2), and DNA aptamer screening methods for selectively binding heavy metals to specifically bind to specific heavy metals may include attaching an amine group to a sensor chip coated with a carboxyl group; And attaching ionized arsenite ions (+3, arsenite) or arsenate (+5, arsenate) to the sensor chip to which the amine group is attached in various forms according to the aqueous solution environment. DNA aptamer selection step that specifically binds heavy metals (see Figure 4). The sensor chip coated with a carboxyl group may be CM5, but is not limited thereto.

In the DNA aptamer screening method of the present invention, the step of screening the DNA aptamer binding to the heavy metal through a heavy metal binding column and SELEX (Systematic Evolution of Ligands by Exponential Enrichment) technique is SELEX round screening having an optimal affinity (affinity) Real time PCR step for; PCR and cloning steps for screening heavy metal binding DNA aptamer candidates in the optimal SELEX round; And DNA sequence determination of the selected heavy metal binding DNA aptamer candidate group.

SELEX method of the present invention is a method for determining the DNA binding sequence of a molecule by selecting and amplifying a DNA having a high binding capacity to a specific molecule in a set of randomly synthesized DNA (Louis et al. 1992. Nature 355, 564- 566).

In the real-time PCR step for SELEX round selection with optimal affinity, real-time PCR of the affinity of ssDNA aptamer eluted in each round for continuous progress and optimal round selection after SELEX up to 10 times As a result of quantitative confirmation using the technique, it was confirmed that the arsenic binding efficiency of the ssDNA aptamer obtained in the ninth round was the highest (see Table 1). PCR and cloning were performed to select heavy metal binding DNA aptamer candidates in the optimal SELEX round, and then the DNA sequences of the selected heavy metal binding DNA aptamer candidates were determined. The sequences of the eight finally selected DNA aptamers are shown in Table 2.

In the DNA aptamer screening method of the present invention, the heavy metal may preferably be arsenic.

The present invention also comprises the steps of attaching an amine group to the surface of the sensor chip coated with a carboxyl group; And attaching ionized arsenite ions (+3, arsenite) or arsenic ions (+5, arsenate) to the sensor chip to which the amine group is attached in various forms according to the aqueous solution environment. (See Figure 4).

Specifically, the arsenic bond sensor chip manufacturing method of the present invention may use a sensor chip coated with a carboxyl group, for example, a sensor chip CM5 (GE Healthcare). The sensor chip CM5 converts the carboxyl group into a more reactive N-hydroxysuccinimide ester, and then treats the surface of the sensor chip CM5 activated with NHS-ester with 1,8-diaminooctane and sodium borate buffer to form amine groups on the chip surface. . This channel 2 of the sensor chip CM5 amine groups are formed, the fugitive ions ionized in various forms according to the aqueous environment on the chip surface by treating the _{Na 2 HAsO 4 (H 3 AsO} 4, H 2 AsO 4 -, HAsO 4 2 - , AsO ₄ ^{3 -)} fixed and ((1 in Fig. 4) Note) the arsenite ions ionize the chip surface was treated in the channel # 3 NaAsO ₂ in various forms according to the aqueous environment (H ₃ AsO _3, H ₂ AsO ^{_{3 -, HAsO 3 2 -,}} AsO 3 3 -) and a fixed reference ((2 in Fig. 4)). The surface of the sensor chip immobilized with arsenic ions (+3, arsenite) or arsenate (+5, arsenate) in various forms according to the aqueous solution environment is inactivated with ethanolamine hydrochloride so that other reagents and ssDNA aptamers Prevents direct sticking.

The present invention also comprises the steps of attaching an amine group to the surface of the sensor chip coated with a carboxyl group;

Attaching ionized arsenite ions (+3, arsenite) or arsenate (+5, arsenate) to the sensor chip to which the amine group is attached in various forms according to the aqueous solution environment;

Optimal arsenic binding DNA aptamer selection step using an arsenic binding sensor chip; And

Arsenic binding measurement for optimal arsenic binding DNA aptamer;

It provides an optimal arsenic binding DNA aptamer selection method comprising a.

As a result of measuring the affinity of the arsenic binding DNA aptamer candidate group in the present invention, it was confirmed that Ars-3 aptamer having the nucleotide sequence of SEQ ID NO: 3 has the best arsenic binding ability (see FIGS. 5 and 6).

Optimum arsenic binding DNA aptamers according to the present invention can be utilized in various environmental fields, specifically arsenic removal and arsenic detection chips contained in colorants, pesticides (pesticides, herbicides, etc.), spent batteries, preservatives, etc. Can be.

The invention also comprises the steps of preparing an arsenic binding DNA aptamer column; And it provides a method for removing arsenic in aqueous solution using the prepared arsenic binding DNA aptamer column. An arsenic binding DNA aptamer column is constructed using the obtained optimal arsenic binding DNA aptamer to provide applicability to the environmentally friendly process of the selected optimal arsenic binding DNA aptamer and industrial applicability. Specifically, the method for preparing the arsenic binding DNA aptamer column of the present invention is modified by arsenic binding DNA aptamer with biotin, and then reacted at 85 ° C. for 5 minutes, and then cooled slowly at room temperature to form a secondary structure. The arsenic binding DNA aptamer thus obtained is injected into a column filled with streptavidin to produce an arsenic binding DNA aptamer column.

In order to determine the arsenic removal rate of the arsenic-binding DNA aptamer column, the arsenic concentration change before and after the arsenic-binding DNA aptamer column of the arsenic aqueous solution was confirmed by using an inductively coupled plasma atomic emission spectroscopy (ICP-AES). do. This step provides the applicability to environmentally friendly heavy metal removal processes utilizing the optimal arsenic binding DNA aptamers and their applicability in various industries.

Hereinafter, the present invention will be described in detail by way of examples. However, the following examples are merely to illustrate the present invention, the content of the present invention is not limited to the following examples.

Example

Example 1: DNA aptamer production process

1.1 Amplification of Random DNA Libraries Using PCR Technique

To prepare ssDNA (single strand DNA) that specifically binds to arsenic, 100bp of template DNA (5'- containing 40 random sequences in the ratio dA: dG: dC: dT = 1.5: 1.15: 1.25: 1) GGTAATACGACTCACTATAGGGAGATACCAGCTTATTCAATT-N40-AGAT TGCACTTACTATCT-3 '; SEQ ID NO: 9) and a pair of primers capable of amplifying it were custom-made to bioneer (Korea). Biotinylated reverse primer: 5'-biotin-AGATTGCACTTACTATCT-3 '(SEQ ID NO: 11)). Any DNA library was amplified using PCR (Biorad, USA). The reaction composition for amplification of the 100bp DNA library by PCR was 5-10 μl of template DNA, 10 μl of 10X PCR buffer, 8 μl of each 2.5 mM dNTP mixture, 1 μl of 25 uM forward primer, 1 μl of 25 uM biotinylated reverse primer. It consists of 20 μl of 5M betaine, 1 μl of Ex Taq polymerase (Takara, Japan) (1 unit / μl) and 49-54 μl of water. PCR reaction conditions were first denatured at 94 ° C. for 5 minutes, followed by 4 cycles of reaction for 30 seconds at 94 ° C., 1 minute at 52 ° C., and 1 minute at 72 ° C., followed by extension at 72 ° C. for 5 minutes. . After the PCR reaction, 2 μl was taken, and the band of the correct size appeared using a 2% agarose gel. The remaining DNA was recovered only DNA using a PCR purification kit (Qiagen, USA).

1.2 ssDNA amplification using asymmetric PCR

Asymmetric PCR was performed to amplify only ssDNA out of the amplified dsDNA using the PCR technique. The asymmetric PCR reaction composition consisted of 50 μl template DNA obtained through 1.1, 10 μl of 10X PCR buffer, 8 μl of 10 mM dNTP mixture, 2 μl of 25 uM forward primer, 1 μl of Ex Taq polymerase (Takara, Japan) (1 unit / μl) And 29 μl of water. Asymmetric PCR reaction conditions were first denatured at 94 ° C. for 5 minutes, followed by 15 cycles of reaction at 30 ° C. at 94 ° C., 1 minute at 52 ° C., and 1 minute at 72 ° C., followed by extension at 72 ° C. for 5 minutes. It was. After the PCR reaction, 2 µl was taken to confirm that the band of the correct size appeared using a 2% agarose gel, and the remaining DNA was added with 1/10 volume of 3M NaOAc (pH4.5) and 2 to 3 volumes of 100% ethanol. After the reaction for 1 hour at -70 ℃, centrifuged for 20 minutes at 13,000 rpm at 4 ℃ to recover only DNA. The recovered DNA is dried in air and then dissolved in 100 µl of distilled water.

1.3 Screening and Recovery of ssDNA

In order to remove dsDNA and ssDNA with biotin from the PCR product amplified by asymmetric PCR and to secure only pure forward ssDNA, 50 μl of streptavidin (Pierce, USA) was added to 100 μl, followed by 2 at 4 ° C. React for hours. After the reaction, the reaction solution was centrifuged for 10 minutes using a centrifugal separator at 4 ° C. and 13,000 rpm, and only the supernatant was recovered. Then, 1/10 volume of 3M NaOAc (pH4.5) and 2 to 3 volumes of 100 Add% ethanol and leave for 1 hour at -70 ℃. After the reaction, centrifuged at 13,000 rpm for 20 minutes at 4 ° C to recover only DNA. The recovered DNA is dried in air and then dissolved in 100 µl of distilled water. 2 μl of the recovered DNA was taken to confirm that a band of the correct size appeared using a 2% agarose gel (see FIG. 1, lane 2). 2 μl of the remaining DNA was taken to treat S1 nuclease, and reacted at 37 ° C. for 1 hour. The recovered ssDNA was completely removed using a 2% agarose gel (see FIG. 1, lane 3).

Example 2: DNA Aptamer Screening for Specific Binding with Arsenic Using SELEX Technique

2.1 Composition of each solution used in SELEX

Resin Equilibrium and Active Solution: 10 mM MOPS Buffer (pH7.5), 1 mM EDTA, 20 mM β-mercaptoethanol

2X DNA Aptamer Selection Solution: 300mM NaCl, 100mM HEPES, 1mM MgCl ₂ , 1mM CaCl ₂

Column Wash Solution: 1X DNA Aptamer Selection Solution, 0.1% tween 20

DNA aptamer elution solution: 150 mM NaCl, 1 mM MgCl ₂ , 1 mM CaCl ₂ , 40 mM β-mercaptoethanol, 45% DMSO, 50 mM HEPES

2.2 Create ssDNA Aptamer for SELEX

To select DNA aptamers that specifically bind to arsenic, 55 μl of distilled water was added to 95 μl of ssDNA, which was prepared and secured, 150 μl of 2X DNA aptamer selection solution was added, and boiled at 95 ° C. for 5 minutes for denaturation. Cool slowly at room temperature to form a stable three-dimensional structure of ssDNA aptamer.

2.3 Arsenic binding column preparation for arsenic binding DNA aptamer screening and screening of ssDNA aptamer using SELEX method

To prepare an arsenic binding column, 4-aminophenylarsine oxide (4PAO) (Sigma, USA) is first dissolved in a DMSO solution at a concentration of 1.7 mg / ml. In order to fix arsenic, 4PAO is coupled to the resin using 0.5 ml of affi-Gel 10 resin (Bio-rad, USA) resin equilibrium and 5 ml of active solution. 4PAO solution dissolved in DMSO was poured onto the activated resin and reacted at 4 ° C. for 1 hour to allow 4PAO to couple to the resin (see FIG. 2). Resin and 4PAO solution were separated and removed by centrifugation, and the resin was washed with 5 × volume of 1X DNA aptamer selection solution to remove the remaining 4PAO solution without binding to the resin. The ssDNA aptamer cooled at room temperature was added to the prepared arsenic binding column and reacted at 4 ° C. for 2 hours. Since ssDNA aptamer that does not specifically adhere to arsenic was removed using 5ml of column washing solution. SsDNA aptamer specifically binding to arsenic eluted only ssDNA aptamer using DNA aptamer elution solution (see FIG. 3A or 3B).

2.4 Nonspecific ssDNA Aptamer Removal with Negative SELEX

In order to remove the ssDNA aptamer adsorbed on the resin other than arsenic, negative SELEX was performed by preparing a pre-column with 4PAO not fixed between 7 and 8 times in SELEX. SsDNA aptamer solution, which was cooled at room temperature, was reacted for 1 hour in a pre-column filled with activated affi-Gel 10 resin using resin equilibrium and an active solution, and ssDNA aptamer solution, which was not bound to the column, was used for 8 times in SELEX. (See FIG. 3A or 3B). SELEX was performed up to 10 times, and the binding condition of SELEX decreased the amount of ssDNA aptamer and reaction time as the number of times increased, and as the number of times increased, the reaction condition was roughened to obtain an aptamer that specifically binds to arsenic as a target substance. Now.

Example 3: Affinity Test Using Real Time PCR Method

After completing SELEX up to 10 times, real-time PCR was performed to quantitatively confirm SELEX progress and affinity of the ssDNA aptamer eluted in each round. First, the ssDNA library and each of the ssDNA aptamers eluted at 8, 9, and 10 rounds were amplified by PCR, and asymmetric PCR was used to secure ssDNA aptamers. The concentration of the prepared ssDNA aptamer was divided into 0 µg, 4 µg and 40 µg, and reacted with the arsenic binding column for 1 hour. Each of the obtained ssDNA aptamers was divided into 10 ⁵ , 10 ⁶ , and 10 ⁷ to proceed with real-time PCR. Real-time PCR was performed using Biorad's iQ SYBR Green Supermix (Bio-rad, USA), and real-time PCR reaction conditions were first denatured at 94 ° C. for 5 minutes, 20 seconds at 94 ° C., 20 seconds at 52 ° C., and 72 ° C. After repeating the reaction for 40 seconds at 20 seconds, the reaction proceeded to the reaction conditions extending for 5 minutes at 72 ℃. According to the real-time PCR, the efficiency of real-time PCR using the 8 and 9 rounds of ssDNA 4 ㎍ for SELEX and 10 ⁶ ssDNA as the template DNA of the real-time PCR was the highest at 84.16% and 85.23%, respectively. The efficiency of the real-time PCR results of the obtained ssDNA is 80.16%, which is inferior to the results of the 9th round, indicating that there is no need to proceed with SELEX. In addition, the ssDNA aptamer obtained in the 9th round based on the real-time PCR results was confirmed that the highest arsenic binding efficiency. Table 1 quantitatively confirms the affinity of each round for the initial ssDNA library and ssDNA eluted at round 8, 9, and 10 of the ssDNA aptamers obtained by performing a total of 10 rounds of SELEX using an arsenic binding column. Table shows the results of performing real-time PCR in order to.

Table 1.

Example 4: Cloning of Arsenic Specific Binding DNA Aptamer Candidates

Forward primers: 5'-GGTAATACGACTCAC TATAGGGAGATACCAGCTTATTCAATT-3 '(SEQ ID NO: 10) and reverse primers: 5'-biotin-AGATTGCACTTACTATCT-3' for 9 rounds of ssDNA aptamers determined to have the highest arsenic binding efficiency by real-time PCR DsDNA was obtained by PCR using (SEQ ID NO: 11). The dsDNA thus obtained was subjected to TA cloning using the T & A cloning kit of RBC. TA cloning was performed by mixing 2 μl of T-vector (25 ng / μl) with 5 μl of PCR product (20 ng / μl), 1 μl of ligase (3 unit), 1 μl of 10 × ligation buffer A, and 1 μl of 10 × ligation buffer B. The reaction was used for 16 hours at ℃. 10 μl of TA cloned ligate was mixed with 200 μl of DH5α and subjected to a heat shock at 42 ° C. for 1 minute and 30 seconds and placed on ice. 800 μl of LB broth was added thereto, and then incubated at 37 ° C. for 1 hour. After incubation, 200 μl of solution was taken and spread on an LB culture plate (LB plate) containing ampicillin (ampicillin, 50 ng / ml), X-gal (50 μg / ml) and IPTG (5 μg / ml). After culturing at 37 ° C. for 15 hours, only white colonies were selected and commissioned by Solgent (Korea) to determine the nucleotide sequence of the aptamer. Table 2 Sequences for eight arsenic binding DNA aptamer candidate groups obtained during the SELEX process using an arsenic binding column are shown.

Table 2.

Example 5: Affinity test of aptamer candidates binding to arsenic using SPR

5.1 Fabrication of arsenic-coated sensor chip CM5 for each affinity test of aptamer candidates combined with arsenic

In order to quantify the affinity between arsenic and the aptamer candidate group obtained in Example 4, the inventors conducted a surface plasmon resonance (SPR) experiment using BIAcore 2000 (BIACORE), an SPR detection system device. .

In order to confirm the affinity between the arsenic and the aptamer candidate group, a sensor chip CM5 (GE Healthcare) whose surface was coated with a carboxyl group was used. The sensor chip CM5 is activated by flowing 0.1M N-hydroxysuccinimide (NHS) and 0.4M N-ethyl-N '-(dimethyl aminopropyl) carbodiimide (EDC) mixture into the chip for 10 minutes at a rate of 5 µl / min. Switched to this better N-hydroxysuccinimide ester. In order to change the surface of the sensor chip CM5 activated with NHS-ester to the amine surface, 0.1M 1,8-diaminooctane and 50mM sodium borate (pH 8.5) buffer were treated for 10 minutes at a rate of 5 μl / min, and the amine was applied to the chip surface. A group was formed. Thus, channel number 2 of the sensor chip CM5 in which the amine group was formed was treated with 1M Na ₂ HAsO ₄ at a rate of 5 μl / min for 10 minutes, and ionized non-acid ions (H ₃ AsO ₄ , ^{_{H 2 AsO 4 -, HAsO 4}} 2-, AsO 4 3 - was fixed) reference ((1 in Fig. 4)). Channel number 3 of the sensor chip CM5 having an amine group was treated with 1M NaAsO ₂ at a rate of 5 μl / min for 10 minutes, and the surface of the chip was ionized in various forms according to the aqueous solution environment (H ₃ AsO ₃ , H ₂ AsO _3). ^-, HAsO ₃ ^{2 -,} and fixed to AsO ₃ ^3-) reference ((2 in Fig. 4)). Sensor chips immobilized with arsenic ions (+3, arsenite) or arsenates (+5, arsenate) in various forms depending on the aqueous environment can be inactivated with 1M ethanolamine hydrochloride (pH8.5) to inactivate other reagents and ssDNA. The aptamer was prevented from sticking directly to the chip, and after each experiment the sensor chip was regenerated with 10 mM EGTA. Rate variables were obtained with the BIA Assessment Program (BIACORE).

5.2 Determination of Affinity of Arsenic Binding DNA Aptamer Candidates

In order to select the aptamer having the best affinity for arsenic, ssDNA aptamer of each candidate group is dissolved in HBS2P buffer (GE Healthcare) and quantified and secured at a concentration of 200 nM. The secured ssDNA aptamer is reacted at 85 ° C. for 5 minutes and then cooled slowly at room temperature to form a stable three-dimensional structure. The fabricated ssDNA aptamer is immobilized with an immobilized sensor chip (channel 1), ionized arsenite ion (+3, arsenite, channel 3) or arsenate ion (+5, arsenate, channel 2) in various forms depending on the aqueous solution environment. Sequential flow to the sensor chip confirmed the affinity between the arsenic binding aptamer candidate group and the arsenite ion (+3, arsenite, channel 3) or arsenate ion (+5, arsenate, channel 2) (see FIGS. 5 and 6). As a result of the experiment using Biacore, Ars-3 binding DNA aptamer candidate group showed the best binding ability with arsenic.

Example 6: Determination of Arsenic Affinity and Structure Determination of Optimal Arsenic Binding DNA Aptamers Obtained by SPR Analysis

The structure of the arsenic binding DNA aptamer Ars-3 obtained through the SPR analysis could be imaged using a DNA mfold program provided by Rensselear polytechnic institute (see FIG. 7A or 7B).

The optimal arsenic-binding DNA aptamer Ars-3 selected in Example 5 was applied to a sensor chip to which nothing was attached, either CM5, arsenite (+3), or arsenate (+5) (arsenate), under kinetic conditions. By injecting DNA aptamers of various concentrations (300 nM, 200 nM, 100 nM, 50 nM, 10 nM), we obtained the dissociation constant (Kd, dissociation) of Ars-3 (see FIG. 8). As a result, the dissociation constant for arsenic binding DNA aptamer Ars-3 was 1.80 ± 0.27 × 10 ⁻⁷ M, which is a very high dissociation constant for small size DNA aptamer measured by SPR analysis.

Example 7: Optimal Arsenic Binding DNA Aptamer Ars-3 and Other Heavy Metals by SPR Analysis

In order to measure the affinity between the arsenic binding DNA aptamer Ars-3 and other heavy metals, a sensor chip SA coated with streptavidin was used. The sensor chip SA activates the sensor chip by flowing 50mM NaOH and 1M NaCl mixture into the chip for 1 minute at a rate of 5µl / min. The aptamer was fixed to streptavidin by gradually flowing a 500 nM arsenic binding aptamer to which biotin was attached at a rate of 5 µl / min for 10 minutes. Various heavy metals, a solution of 1M to do so, the sensor chip production _{(FeCl 2, MnCl 2, NiCl} 2, CoSO 4, ZnCl 2, CaCl 2, MgCl 2, CdCl 2, NaAsO 2, Na 2 HAsO 4, CuSO ₄ ) was reacted for 2 minutes at a rate of 20 μl / min. As a result, Ars-3 DNA aptamers reacted optimally with arsenic Na ₂ HAsO ₄ and reacted very little with other heavy metals (see FIG. 9).

Example 8: Construction of Arsenic Binding DNA Aptamer Column Using Optimum Arsenic Binding DNA Aptamer Ars-3

The optimal arsenic binding DNA aptamer discovered above was modified with biotin, reacted at 85 ° C. for 5 minutes, and then cooled slowly at room temperature to form a secondary structure. The arsenic binding DNA aptamer thus obtained was injected into a column filled with streptavidin to react the arsenic binding DNA aptamer with streptavidin, and then distilled water was removed to remove DNA aptamers not completely bound to streptavidin. 10 ml was used to wash the arsenic binding DNA aptamer column. As a control, a column filled with streptavidin was used (see FIG. 10).

Example 9 Arsenic Removal and Removal Efficiency Measurement Using Arsenic Binding DNA Aptamer Column

An aqueous arsenic solution containing 100 ppm NaAsO ₂ , 100 ppm Na ₂ HAsO ₄ , 50 ppm NaAsO ₂ , and 50 ppm Na ₂ HAsO ₄ was flowed into the prepared arsenic binding DNA aptamer column and the streptavidin column used as a control. The arsenic concentration change contained in the aqueous solution was measured. Arsenic concentration change in aqueous solution was measured using ICP-AES (see FIGS. 11 and 12). The change in the concentration of arsenic in the aqueous solution is the arsenic concentration of the aqueous solution containing arsenic, the arsenic concentration change value of the aqueous solution passed through the streptavidin column (before the control column, Fig. 10A (a); after passing the control column, A- (b) of FIG. 10 and the arsenic concentration change in the aqueous solution passing through the arsenic binding DNA aptamer column (B- (c) of FIG. 10 before passing the arsenic binding DNA aptamer column; arsenic binding DNA aptamer column) After the passage, the concentration of arsenic changed by ICP-AES was measured by dividing by B- (d) of FIG. 10 and the like. As a result of measuring the change in arsenic concentration in the sample using ICP-AES, the control column containing streptavidin did not remove any arsenic in the sample, whereas arsenic prepared by attaching arsenic binding DNA aptamer to streptavidin The binding DNA aptamer column was confirmed to have an efficiency of removing 100% of arsenic in the sample (see FIGS. 11 and 12).

Figure 1 shows amplification of the random DNA aptamer using PCR and asymmetric PCR techniques, and selectively recovers only ssDNA using streptavidin beads and S1 nuclease treatment of the recovered ssDNA. It shows the result confirmed through.

Lane 1: 100 bp DNA marker

Lane 2: amplification of DNA aptamer using PCR technique, followed by selective recovery of ssDNA using streptavidin beads.

Lane 3: S1 nuclease treatment of ssDNA recovered using streptavidin beads.

Figure 2 shows the principle utilized in the arsenic binding column fabrication process to select the DNA aptamer specifically binding to arsenic.

3A to 3B show experimental results that the DNA aptamer specifically binding to arsenic can be selected using a column to which arsenic is bound.

A: Attached and eluted the DNA aptamer labeled with cy5 to the arsenic binding column and confirmed by fluorescence scanner.

B: After attaching and eluting a DNA aptamer labeled with cy5 to the column which did not bind arsenic, it was confirmed using a fluorescence scanner.

4 shows amine groups formed on the surface of the sensor chip CM5 and ionized non-acid ions (+5, arsenate, panel (1)) or arsenite ions (+3, arsenite, panel (2)) in various forms according to the aqueous solution environment. It shows the process of fixing.

5 is a result of confirming the most affinity DNA aptamer obtained by flowing the obtained arsenic binding DNA aptamer candidate group to the sensor chip CM5 in which ionized non-acid ions (+5, arsenate) are fixed in various forms according to the aqueous solution environment. .

6 is a result of confirming the most affinity DNA aptamer by flowing the obtained arsenic binding DNA aptamer candidate group to the sensor chip CM5 in which ionized arsenic ions (+3, arsenite) are fixed in various forms according to the aqueous solution environment. .

Figure 7 shows the secondary structure of the Ars-3 DNA aptamer shown to have the best binding to ions (+5, arsenate) or arsenite (+3, ions) ionized in various forms depending on the aqueous environment.

Figure 8 shows the change in the binding force of the ionized non-acid ions (+5, arsenate) in various forms according to the aqueous solution environment according to the concentration of Ars-3 DNA.

Figure 9 is the coupling efficiency and the Arsenic appears to be the best Ars-3 DNA aptamer and a variety of other heavy metals _{(FeCl 2, MnCl 2, NiCl} 2, CoSO 4, ZnCl 2, CaCl 2, MgCl 2, CdCl 2, NaAsO 2, Na ₂ HAsO ₄ , CuSO ₄ ) is the result of measuring the binding force.

FIG. 10 is a diagram illustrating an experimental design for confirming arsenic removal in an aqueous solution by using the Ars-3 DNA aptamer that optimally binds to arsenic and constructs an arsenic binding DNA aptamer column.

FIG. 11 is a graph of measuring arsenic concentration change in an aqueous solution using ICP-AES to confirm the removal of arsenic in an aqueous NaAsO ₂ solution using the arsenic binding DNA aptamer column. Arsenic concentration was divided into 50 ppm and 100 ppm, and the experiment was conducted. The NaAsO ₂ aqueous solution, the NaAsO ₂ aqueous solution passed through the streptavidin column, and the NaAsO passed through the arsenic binding DNA aptamer column having the DNA aptamer attached to the streptavidin in NaAsO ₂ solution if the result of each divided measuring the concentration of the _second aqueous solution, streptavidin both before and after passing the aqueous solution through the avidin column but the same arsenic concentrations to 50 ppm or 100ppm is detected, through the non-small binding DNA aptamer column It shows 100% removal of arsenic.

12 is Na ₂ HAsO ₄ using the arsenic binding DNA aptamer column In order to confirm the arsenic removal rate in the aqueous solution state, the arsenic concentration change in the aqueous solution was measured using ICP-AES. Arsenic concentrations of 50 ppm, was the experiment divided by 100 ppm, Na ₂ HAsO ₄ aqueous solution, streptavidin-one Na ₂ HAsO passed through the column _4, an aqueous solution, which attach the DNA aptamer arsenic bound to streptavidin-DNA aptamer column As a result of dividing and measuring the concentrations of the Na ₂ HAsO ₄ aqueous solution passed through the solution, 50 ppm or 100 ppm of arsenic concentration was detected before and after the passage through the streptavidin column, but passed through the arsenic binding DNA aptamer column. In one case, 100% of the arsenic contained in the Na ₂ HAsO ₄ aqueous solution was removed.

<110> KIM, Yang-Hoon          MIN, Jiho          OH, Suk Jung <120> DNA aptamer specifically binding to arsenic and uses <130> PN08128 <160> 11 <170> KopatentIn 1.71 <210> 1 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> DNA aptamer <400> 1 accatcgcgg aagtccagtc tgccatcaaa atccgaagtg 40 <210> 2 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> DNA aptamer <400> 2 cacgcgttca acccccggaa tttagcaata gcagattacg 40 <210> 3 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> DNA aptamer <400> 3 ttacagaaca accaacgtcg ctccgggtac ttcttcatcg 40 <210> 4 <211> 39 <212> DNA <213> Artificial Sequence <220> <223> DNA aptamer <400> 4 ttccgctagg ggaacatgat caacatggac caagtaaac 39 <210> 5 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> DNA aptamer <400> 5 cagtcagaat ccgggcttac acccatttgt ttattgtgca 40 <210> 6 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> DNA aptamer <400> 6 caatctaagc gaaccgcgtg cagaccaatt actcgcgcta 40 <210> 7 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> DNA aptamer <400> 7 atgcaaaccc ttaagaaagt ggtcgtccaa aaaaccattg 40 <210> 8 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> DNA aptamer <400> 8 tggggattgt acgtacacca ccaattcaca cgcgagtatg 40 <210> 9 <211> 100 <212> DNA <213> Artificial Sequence <220> <223> template DNA <400> 9 ggtaatacga ctcactatag ggagatacca gcttattcaa ttnnnnnnnn nnnnnnnnnn 60 nnnnnnnnnn nnnnnnnnnn nnagattgca cttactatct 100 <210> 10 <211> 42 <212> DNA <213> Artificial Sequence <220> <223> Forward primer <400> 10 ggtaatacga ctcactatag ggagatacca gcttattcaa tt 42 <210> 11 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer <400> 11 agattgcact tactatct 18  

Claims (21)

DNA aptamer specifically binding to arsenic (As) having at least one nucleotide sequence of the base sequence of SEQ ID NO: 1 to SEQ ID NO: 8. delete delete The DNA aptamer according to claim 1, wherein the DNA aptamer is single stranded DNA. Arsenic removal or detection composition comprising a DNA aptamer having at least one nucleotide sequence of the base sequence of SEQ ID NO: 1 to SEQ ID NO: 8 as an active ingredient. delete A microarray for detecting arsenic having a substrate on which a DNA aptamer having at least one nucleotide sequence of SEQ ID NO: 1 to SEQ ID NO: 8 is immobilized. delete delete Arsenic detection kit comprising a DNA aptamer having at least one nucleotide sequence of the base sequence of SEQ ID NO: 1 to SEQ ID NO: 8. delete Contacting a sample suspected of containing arsenic with a DNA aptamer having at least one nucleotide sequence of SEQ ID NO: 1 to SEQ ID NO: 8 and detecting the binding of the arsenic to the DNA aptamer; A method for detecting arsenic in a sample. delete Contacting a sample suspected of containing arsenic with a DNA aptamer having at least one nucleotide sequence of SEQ ID NO: 1 to SEQ ID NO: 8 to form an arsenic and DNA aptamer complex and removing the complex A method for removing arsenic in a sample comprising the step. delete delete delete delete delete delete Preparing a DNA aptamer column having a nucleotide sequence of SEQ ID NO: 3 that binds to arsenic; And removing arsenic in the aqueous solution using the prepared arsenic binding DNA aptamer column.

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