WO2009041776A2

WO2009041776A2 - Dna aptamer binding to oxytetracycline with specificity and production method thereof

Info

Publication number: WO2009041776A2
Application number: PCT/KR2008/005696
Authority: WO
Inventors: Man Bock Gu; Javed Hossain Niazi K.M.; Yeon Seok Kim; Yoonjin Kim; Su Jin Lee
Original assignee: Korea University Industrial & Academic Collaboration Foundation
Priority date: 2007-09-27
Filing date: 2008-09-25
Publication date: 2009-04-02
Also published as: KR20090032276A; KR100930973B1; WO2009041776A3

Abstract

Provided is a DNA aptamer which binds specifically to Oxytetracycline (OTC). More particularly, there are provided a DNA aptamer which can bind specifically to OTC selected from a random DNA library, a method for producing the same, and a method for detecting or removing OTC using the same. DNA aptamers showing specifically high affinity for the antibiotics OTC were selected from a random DNA pool using the FluMag-SELEX process, a modification of the SELEX process. The selected DNA aptamers were confirmed to have strong binding affinity for OTC. Accordingly, the DNA aptamers provided herein may be used to effectively detect antibiotics remaining in drinking water sources, sewage, foods, human bodies, etc. Further, the DNA aptamers may be used to selectively remove a target substance from a sample containing a trace amount of antibiotics.

Description

[DESCRIPTION] [Invention Title]

DNA aptamer binding to Oxytetracycline with specificity and production method thereof

[Technical Field]

The present invention relates to a DNA aptamer which binds specifically to Oxytetracycline (OTC), more particularly to a DNA aptamer which can bind specifically to OTC selected from a random DNA library, a method for producing the same, and a method for detecting or removing OTC using the same.

[Background Art]

Oxytetracycline (OTC) is a member of the widely used tetracycline antibiotics group. It is used as feed additives or growth promoters for animals. It is commonly used in fish farms for rockfish, flatfish, etc. or added to prevent fishery products or stock farm products from infections. However, the problem is that OTC remaining in the remnants of animal feeds or in the feces may flow into soil or water and affect humans through various routes. Especially, it may turn bones or teeth into a yellowish brown color in pregnant women or young children. Further, OTC is known to be associated with a high risk of birth defect because it delays skeletal growth of the fetus.

Long-term intake of fishery or stock farm products containing OTC or other antibiotics may result in reduced immunity or other side effects because of acquired resistance to the antibiotics. In other words, excessive use of antibiotics in animals may turn common food poisoning bacteria into antibiotics-resistant super bacteria. Then, stronger antibiotics are needed to treat food poisoning. In order to prevent this vicious cycle, development of a method for detecting antibiotics remaining in food is necessary, let alone abstaining from abuse thereof.

Aptamers are single-stranded DNA or RNA structures having high specificity and affinity for a specific target. Aptamers provide much superior affinity for the target as compared to antibodies commonly used as sensing material in the field of sensor and provide superior thermal stability. Since they can be synthesized in vitro, they are favorable in terms of cost because there is no need to inject antigens into animals to obtain antibodies. Further, with no limitation on target substances, aptamers can be synthesized for various targets, including biomolecules such as proteins and amino acids, small organic substances such as environmental hormones and antibiotics, bacteria or the like. Recently, the characteristic of the aptamers of specifically binding to target substances has led to active researches on development of new medicines, drug delivery systems, biosensors, and the like. Accordingly, the aptamers are well suited for the detection of a trace amount of antibiotics and may be applied to detect other specific materials through nanobiotechnology.

In existing antibiotics detection techniques, different test solutions are prepared for substances containing different antibiotics and the antibiotics are extracted, purified and quantitated by liquid chromatography. In contrast to aptamers, which can specifically bind to OTC in any substance and enable detection, the existing methods provide only a limited number of test methods depending on substances and the procedure of preparing the test solutions is complicated. Additionally, exact quantitation is difficult, because OTC may be lost during extraction or purification. Differently from our technology, which enables the detection of low molecular weight medicines or antibiotics, most existing aptamer developments are targeting proteins for the development of biosensors for medical diagnosis or new medicines. These aptamers binding specifically to low molecular weight harmful substances such as residual drugs, antibiotics and environmental hormones can be applied not only to samples from drinking water sources and rivers but also to the detection of harmful substances in human or other organisms for medical applications. In contrast, the existing antibiotics detection techniques are applicable only to food or water.

The inventors of the present invention have researched to overcome the problems associated with the existing techniques. As a result, we developed a DNA aptamer as a nucleotide structure showing specifically high affinity for OTC, a kind of antibiotics. Further, we prepared a composition comprising the DNA aptamer capable of binding specifically to OTC and confirmed that it can be usefully used to detect antibiotics from foods, drinking water sources, sewage, human bodies, and the like.

[Disclosure] [Technical Problem]

Accordingly, an object of the present invention is to provide a DNA aptamer showing specifically high affinity for Oxytetracycline (OTC) and a production method thereof.

Another object of the present invention is to provide a composition for the detection or removal of OTC comprising the DNA aptamer.

Still another object of the present invention is to provide a method for the detection or removal of OTC using the DNA aptamer. [Technical Solution]

In an aspect, the present invention provides a DNA aptamer which is capable of binding specifically to Oxytetracycline (OTC). In the present invention, the DNA aptamer which is capable of binding specifically to OTC was selected by the SELEX (Systematic Evolution of Ligands by Exponential Enrichment) process. The SELEX process used in the present invention refers to a procedure of identifying DNA sequence by using DNAs or RNAs with high affinity for a particular molecule, which are selected from a random pool of synthetic DNAs or RNAs [Louis C. Bock, Linda C. Griffin, John A. Latham, Eric H. Vermaas, John J. Toole. 1992. Selection of single-stranded DNA molecules that bind and inhibit human thrombin. Nature 355, 564-566].

The DNA aptamer used in the present invention may be a DNA aptamer having any base sequence that specifically binds to OTC, being selected the SELEX process. Preferably, it may have a base sequence of any of SEQ ID NOS: 1-15.

In another aspect, the present invention provides a production method of a DNA aptamer which is capable of binding specifically to OTC, the method comprising: a) reacting OTC with magnetic beads in borate buffer solution to induce covalent bonding; b) mixing a pool of DNAs having PCR primer regions at both ends and 30-50 bp of random nucleotides therebetween with the covalently bonded OTC-magnetic beads at room temperature in buffer solution to induce binding; c) separating the DNAs bound to the OTC-magnetic beads using a magnet; d) separating the DNAs from the separated OTC-magnetic beads; and e) carrying out PCR using a pair of primers complementary to the PCR primer regions to amplify the DNAs specifically binding to OTC.

In the production method of the present invention, the covalent bonding in a) may be a covalent bonding between the amino groups of OTC and the tosyl groups of the magnetic beads.

The production method of the present invention may further comprise, after a), deactivating the tosyl groups of the magnetic beads not bound to OTC with ethanolamine at 40-50⁰C for 6-18 hours.

In the production method of the present invention, the separation of DNAs in d) may be carried out by treating the OTC-magnetic beads at 70-90⁰C for 3-10 minutes.

The production method of the present invention may further comprise in e) carrying out PCR using a pair of primers, one of which is labeled with fluorescein, followed by separating single-stranded DNAs modified by fluorescein by electrophoresis.

In another aspect, the present invention provides a composition for detection or removal of OTC comprising a DNA aptamer which is capable of binding specifically to OTC. The composition may be included in an apparatus or kit for detection or removal of OTC.

In the composition of the present invention, the DNA aptamer may have a base sequence of any of SEQ ID NOS: 1 -15.

In another aspect, the present invention provides a use of a DNA aptamer which is capable of binding specifically to OTC for detection or removal of OTC.

The DNA aptamer of the present invention is for detecting OTC, which is the most commonly used antibiotics, exceeding the allowed limit. Since even a very small amount of antibiotics remaining in foods or environment may affect humans, the final consumer in the food chain, through a variety of routes including biomagnification, it is necessary to detect and remove the antibiotics extensively used for fishery products and stock farm products. For the detection of OTC, the DNA aptamer of the present invention may be used in any form. For instance, the DNA aptamer of the present invention may be fixed on magnetic beads and bound with OTC. Then, the resultant DNA aptamer-OTC complex may be separated using a magnet, and only OTC may be separated from the complex. In addition to the method described in this description, a sensor comprising the DNA aptamer of the present invention linked by a linker may be used to detect OTC from a sample, for example.

Further, only OTC may be selectively removed from a sample using the composition of the present invention. For instance, the magnetic beads on which the DNA aptamer is fixed may be packed in a column and then the OTC-containing sample may be passed therethrough in order to selectively remove OTC.

[Description of Drawings]

Description will now be made in detail with reference to certain example embodiments illustrated in the accompanying drawings which are given hereinbelow by way of illustration only and thus are not limitative, wherein:

Fig. 1 shows the chemical structure of Oxytetracycline (OTC);

Fig. 2 schematically illustrates the method of designing a nucleotide structure which specifically binds to OTC;

Fig. 3 shows the percentage of ssDNA eluted from the OTC-fixed magnetic beads of each selection;

Figs. 4-18 schematically illustrate the secondary structure (base sequence) of 15 nucleotide structure aptamers which specifically bind to OTC, predicted by the m-fold program;

Figs. 19-23 show the binding curve of the 5 OTC-aptamers having the strongest binding affinity with OTC;

Fig. 24 is a graph showing that the aptamer of the present invention exhibits selective specificity only for OTC and does not exhibit affinity for other chemicals with similar structure.

[Best Mode]

Hereinafter, the present invention will be described in more detail.

1) Fixation of Oxytetracvcline (OTC) on magnetic beads

Covalent bonding is formed between the amino groups of OTC and the tosyl groups of magnetic beads. To this end, OTC and magnetic beads are reacted in buffer solution. The OTC-bound magnetic beads can be separated using a magnet. The beads are washed with the same buffer solution in order to remove OTC not bound to the beads.

2) Development of DNA aptamer binding specifically to OTC

The SELEX (Systematic Evolution of Ligands by Exponential Enrichment) process is widely employed to develop an aptamer specific for a target substance. In the present invention, the FluMag-SELEX process, which is a modification of the SELEX process, is used to develop a DNA aptamer which binds specifically to OTC. In the FluMag-SELEX process used in the present invention, a fluorescence labeled primer is used in the DNA amplification step such that only the band exhibiting fluorescence may be taken in the following PCR product separation process (dsDNA → ssDNA) using PAGE. The DNAs binding to the target and those not binding thereto are separated using a magnet. In other existing SELEX procedures, radioactive labeling, capillary electrophoresis, membrane filtration, or the like are employed. But, they are costly and difficult for handling. Further, chromatography, affinity column, or the like are used for the separation of the PCR product, but they are expensive and ineffective [R. Stoltenburg, C. Reinemann, B. Strehlitz (2005) Anal. Bioanal. Chem. 383: 83-91].

First, a DNA pool consisting of 76 random nucleotides is prepared. The DNA pool is mixed with OTC-fixed magnetic beads in buffer solution. Then, the DNAs not binding with OTC are removed using a magnet. After separating OTC-bound DNAs from the magnetic beads by thermal treatment, the DNAs are obtained by elution through ethanol precipitation.

PCR is carried out in order to amplify the DNAs binding specifically to OTC. The PCR product is purified and subjected to electrophoresis using polyacrylamide gel including a high concentration of urea. Two split single-stranded DNAs are obtained. After gel extracting an adequate DNA band, the DNAs are obtained by ethanol precipitation. Thus obtained DNA pool is mixed with the original solution of OTC-fixed magnetic beads. This procedure is repeated to obtain a DNA pool in which at least 90% of DNAs are bound to OTC. The DNA pool is cloned using a TOPO cloning kit and the DNAs obtained from the resulting colony is subjected to base sequence analysis. As a result, 15 different nucleotide structures binding specifically to OTC are obtained.

[Mode for Invention] Hereinafter, reference will be made in detail to various embodiments of the present invention referring to examples. However, the following examples are for the purpose of illustration only and they are not intended to limit the scope of the present invention.

Example 1 : Binding of OTC to magnetic beads

Covalent bonding was formed between the amino groups of Oxytetracycline (OTC, Sigma Co.) and the tosyl groups of magnetic beads (M-280 tosyl-activated magnetic beads, Dynal Biotech ASA, Norway). To this end, 10 mM OTC and 0.2^10⁹ magnetic beads were reacted in buffer solution (0.1 M borate buffer, pH 10.5) at 45⁰C for 24 hours. OTC-bound magnetic beads (OTC-magnetic beads) could be separated using a magnet. OTC not binding to the beads was removed by washing with the same buffer solution. After washing, the OTC-magnetic beads were reacted in 0.5 M ethanolamine solution (pH 8.0) at 45⁰C for 12 hours to deactivate the tosyl groups not binding to OTC. Because the presence of active tosyl groups may result in nonspecific binding through various intermolecular interactions, e.g. van der Waals bonding, hydrophobic interaction, etc., the active groups were blocked using ethanolamine, which is known to be substantially inert. Following several times of washing, the magnetic beads were mixed with buffer solution (0.1 M borate, pH 9.5) and stored at 4⁰C.

Example 2: Preparation of DNA pool having random base seguence

A 76-mer DNA pool having PCR primer regions at both ends and 40 bp of random nucleotides therebetween was prepared as follows. The DNA pool used in the present invention was chemically synthesized by Genotech Inc. Korea (5'-CGTACGGAATTCGCTAGC-N40-GGATCCGAGCTCCACGTG-3').

Example 3: Selection of DNA aptamer binding to OTC The random DNA pool prepared in Example 2 was mixed with magnetic beads in which 9.35 μmol OTC was fixed in buffer solution (20 mM Tris-CI buffer, pH 7.6, containing 100 mM NaCI, 2 mM MgCI₂, 5 mM KCI, 1 mM CaCI₂ and 0.02% Tween 20) at room temperature for 30 minutes. The DNAs not binding to OTC were removed using a magnet. The DNAs weakly binding to OTC were removed by washing 5 times with the buffer solution. The DNAs binding to OTC were eluted by placing the tube containing the mixture at 8O⁰C for 5 minutes, followed by ethanol precipitation. The amount of thus obtained OTC-bound DNA was measured. Fig. 3 shows the percentage of ssDNA eluted from the OTC-fixed magnetic beads of each selection.

Example 4: Preparation of DNA aptamer pool capable of binding to OTC

In order to amplify the DNAs specifically binding to OTC, PCR was carried out using known primer regions. Because the PCR product is double-stranded DNAs, they were separated into single strands as follows. One of the primers was fixed with fluorescein: forward (APTFf) 5'-fluorescein-CGTACGGAATTCGCTAGC-3', reverse (APTR) 5'-CACGTGGAGCTCGGATCC-3'.

The PCR product was purified using a purification kit and polyacrylamide gel electrophoresis was carried out in order to separate the double-stranded DNAs into single strands. 6 M urea and 20% formamide were included in 10% polyacrylamide gel such that two bands are formed following electrophoresis. During electrophoresis, the double-stranded DNA is modified. As a result, the fluorescein-bound DNA strands is positioned upward and the fluorescein-free DNA strands is positioned downward. The fluorescein-bound DNA band was cut and subjected to gel extraction. Then, separated DNAs were obtained by ethanol precipitation. Thus obtained DNA pool was mixed again with the original solution containing the OTC-fixed magnetic beads. This procedure is schematically illustrated in Fig. 2. This procedure (FluMag-SELEX process) is called one selection. Through 7 selections, a DNA pool in which at least 90% of DNAs are bound to OTC was obtained. After 2nd and 4th selections, counter-selection was carried out using ethanolamine and tetracycline, respectively, in order to block the DNAs binding non-specifically and selecting only the DNAs binding specifically to OTC with high affinity. In Fig. 3, the percentage of ssDNA eluted from the OTC-fixed magnetic beads of each selection is given. Thus obtained DNA pool was cloned using a TOPO cloning kit and the DNAs obtained from the resulting colony was subjected to base sequence analysis. As a result, 15 different nucleotide structures binding specifically to OTC were obtained.

Example 5. Base sequence analysis and OTC affinity measurement for 15 DNA aptamers

Base sequence analysis result for the 15 different DNAs binding specifically to OTC with high affinity is given in the following Table 1. Further, Figs. 4-18 schematically illustrate the secondary structure of 15 nucleotide structure aptamers which specifically bind to OTC, predicted by the m-fold program.

[Table 1] Base sequence of 15 different DNAs binding specifically to OTC with high affinity

From the 15 DNA aptamers, the 5 aptamers with the highest affinity for OTC were selected as follows.

50 μM OTC and each OTC aptamer (153 nM) were reacted in buffer solution (20 mM Tris-CI buffer, pH 7.6, containing 100 mM NaCI, 2 mM MgCI₂, 5 mM KCI, 1 mM CaCI₂ and 0.02% Tween 20) at room temperature for 30 minutes. The product was put in a Microcon filter (YM 10) (Millipore Co., USA) and centrifuged at 30,000 x g for 11 minutes. Base on the concentrations of the OTC binding with the aptamer and remaining on the filter and the OTC not binding with the aptamer and passing through the filter determined by absorbance measurement at 375 nm, the 5 aptamers with the highest affinity for OTC were selected (No. 2, No. 4, No. 5, No. 8 and No. 20).

Example 6: Analysis of binding affinity to OTC of 5 DNA aptamers

The binding affinity of the 5 aptamers with the highest affinity for OTC was analyzed. 50 μM OTC and the aptamers at different concentrations ranging from 0 to 500 nM were reacted in buffer solution (2OmM Tris-CI buffer, pH 7.6, containing 100 mM NaCI, 2 mM MgCI₂, 5 mM KCI, 1 mM CaCI₂ and 0.02% Tween 20) at room temperature for 30 minutes. For the measurement of the dissociation constant, the product was put in a Microcon filter (YM 10) and centrifuged at 30,000 x g for 11 minutes. Absorbance of the OTC binding with the aptamer and remaining on the filter and the OTC not binding with the aptamer and passing through the filter was measured at 375 nm.

The concentration of OTC bound to each aptamer was plotted using SigmaPlot 8.0 based on nonlinear regression and one-site saturation ligand binding. The equation y = B_max x X/K_d + X (y = degree of saturation, B_max = maximum binding sites, K_d = dissociation constant, X = number of unbound aptamers) was used.

The result is given in the following Table 2. No. 4 (K_d = 9.613 nM) and No. 5 (K_d = 11.13 nM) exhibited very strong binding affinity for OTC. Binding curves for the respective aptamers are given in Figs. 19-23.

[Table 2] Dissociation constant (K_d) values of 5 aptamers

Example 7: Confirmation of OTC binding specificity of 5 DNA aptamers In order to confirm that the OTC aptamers bind specifically only to OTC, OTC analogs tetracycline and doxycycline were used as control compounds. Because counter-selection was carried out following the 2nd and 4th selections, as described in Example 4, it is believed that nonspecific binding is almost nonexistent. However, test was carried out in order to demonstrate that the aptamers bind specifically only to OTC, and not to other chemicals.

First, tetracycline was fixed on magnetic beads. Then, the tetracycline-fixed magnetic beads and the 5 OTC aptamers (Nos. 2, 4, 5, 8 and 20) were reacted, respectively, in buffer solution (20 mM Tris-CI buffer, pH 7.6, containing 100 mM NaCI, 2 mM MgCI₂, 5 mM KCI, 1 mM CaCI₂ and 0.02% Tween 20) at room temperature for 30 minutes. The aptamers not binding to tetracycline were separated using a magnet. The DNAs binding to tetracycline were separated by heat treatment, followed by the measurement of the DNAs eluted from the magnetic beads by ethanol precipitation. The same procedure was carried out for doxycycline. As a result, binding to tetracycline was 3.2% (No. 4) and 2.5% (No. 5), and binding to doxycycline was 1.7(No. 4) and 0% (No. 5). The five OTC aptamers (Nos. 2, 4, 5, 8 and 20) including No. 4 and No. 5 were proven to bind specifically only to OTC. The result is given in Fig. 24.

[Industrial Applicability]

As described, the DNA aptamer of the present invention, which is capable of binding specifically to Oxytetracycline (OTC), may be used to detect a trace amount of antibiotics remaining in water or foods more sensitively. Further, the DNA aptamer of the present invention may be employed in a method or apparatus for removing antibiotics to selectively remove a target substance from a sample including a trace amount of antibiotics. Accordingly, it may be utilized to protect humans from the emergence of antibiotics-resistant bacteria, biomagnification and other phenomena, which may result from the presence of a trace amount of antibiotics in foods or environment.

Claims

[CLAIMS] [Claim 1 ]

A DNA aptamer which is capable of binding specifically to Oxytetracycline (OTC).

[Claim 2]

The DNA aptamer as set forth in claim 1 , wherein the DNA aptamer has a base sequence of any of SEQ ID NOS: 1-15.

[Claim 3]

A production method of a DNA aptamer which is capable of binding specifically to OTC, comprising: a) reacting OTC with magnetic beads in borate buffer solution to induce covalent bonding; b) mixing a pool of DNAs having PCR primer regions at both ends and 30-50 bp of random nucleotides therebetween with the covalently bonded OTC-magnetic beads at room temperature in buffer solution to induce binding; c) separating the DNAs bound to the OTC-magnetic beads using a magnet; d) separating the DNAs from the separated OTC-magnetic beads; and e) carrying out PCR using a pair of primers complementary to the PCR primer regions to amplify the DNAs specifically binding to OTC.

[Claim 4]

The production method as set forth in claim 3, wherein the covalent bonding in a) is a covalent bonding between the amino groups of OTC and the tosyl groups of the magnetic beads.

[Claim 5]

The production method as set forth in claim 3, which further comprises, after a), deactivating the tosyl groups of the magnetic beads not bound to OTC with ethanolamine at 40-50⁰C for 6-18 hours.

[Claim 6]

The production method as set forth in claim 3, wherein the separation of DNAs in d) is carried out by treating the OTC-magnetic beads at 70-90⁰C for 3-10 minutes.

[Claim 71

The production method as set forth in claim 3, which further comprises in e) carrying out PCR using a pair of primers, one of which is labeled with fluorescein, followed by separating single-stranded DNAs modified by fluorescein by electrophoresis.

[Claim 8]

A composition for detection of OTC comprising a DNA aptamer which is capable of binding specifically to OTC.

[Claim 9] A use of a DNA aptamer which is capable of binding specifically to OTC for detection of OTC.

[Claim 10]

A composition for removal of OTC comprising a DNA aptamer which is capable of binding specifically to OTC.

[Claim 11 ]

A use of a DNA aptamer which is capable of binding specifically to OTC for removal of OTC.