WO2012108669A2

WO2012108669A2 - Uranyl ion specific dna aptamer

Info

Publication number: WO2012108669A2
Application number: PCT/KR2012/000889
Authority: WO
Inventors: 하상수; 김지수
Original assignee: 경희대학교 산학협력단
Priority date: 2011-02-08
Filing date: 2012-02-07
Publication date: 2012-08-16
Also published as: WO2012108669A3; KR101238297B1; KR20120090477A

Abstract

Provided is a uranyl ion specific DNA aptamer. Also provided is a uranyl ion specific DNA aptamer which is bound to a solid support. Also provided is a method for isolating uranyl ions from a sample by using the uranyl ion specific DNA aptamer. The DNA aptamer of the present invention binds to uranyl ions with high specificity, such that uranium can be efficiently extracted from marine and other such environments.

Description

Uranyl ion-specific DNA Aptamers

The present invention relates to metal-specific aptamers. More specifically, the present invention relates to uranyl ion-specific DNA based aptamers.

Chelation of metals from water is important for environmental protection and resource recovery. In particular, metal-specific chelating resins are much more effective than simple ion exchange resins in metal chelation because they exhibit higher selectivity and greater complexation constants for target metals and can be used for drinking or wastewater treatment and seawater. It is because it is useful in practical use, such as extraction of a target metal ion from the. In view of this, uranium, so that almost four billion tons in the world nuclear be used for about 6 mannyeon present in a concentration of about 3 ppb as tricarboxylic Bona sat complex of mainly uranyl ion from the sea water, uranyl ion (UO _{² 2} the design of an effective host molecule for ⁺⁾ is, IAEA (International Atomic Energy Agency), the data released in May of 2008 (global uranium reserves 4,740,000 tons and estimated reserves as 10.55 million tons this year 6.4 million tonnes world Considering the annual usage, the amount that can be used for about 70 years) is linked to the economic importance of selective extraction of uranium from seawater. However, systems developed so far for uranyl ion bonding have been found to lack the specificity required for uranyl ions due to interference of other metal ions. As a result, studies on the selective molecular recognition of uranil ions using macrocyclic host molecules including crown ethers and calixarenes have been carried out, but such macrocyclic host molecules require much labor to synthesize. And undesired because of the deformation that is difficult to bond to the solid support.

Aptamers, on the other hand, are non-naturally occurring structured oligonucleotides capable of binding DNA, RNA, small molecules, peptides, proteins, and the like. In general, aptamers are produced by an in vitro selection process called systemic evolution of ligands by exponential enrichment (SELEX), and are used in many applications such as specific detection and target-specific delivery of proteins, small molecules, and the like. It is used in bioanalytical applications.

The present inventors completed the present invention by developing an aptamer capable of binding with high specificity to uranil ions while studying to find uranil ion-specific materials that can be used to effectively extract uranium from seawater. It was.

It is an object of the present invention to provide uranyl ion-specific DNA based aptamers conjugated to uranyl ion-specific DNA based aptamers and solid supports.

It is also an object of the present invention to provide a method for separating uranil ions from a sample that is believed to contain uranil ions using an uranil ion-specific DNA based aptamer.

In order to achieve the above object, the present invention provides a uranil ion (UO _{2 2} ⁺ )-specific DNA-based aptamer (HS-DNA 1) having the following structure and HS-DNA 1 conjugated to a solid support to provide.

The uranil ion-specific DNA-based aptamers of the invention are 5'-CTGCA GAATT CTAAT ACGAC TCACT ATAGG AAGAG ATGGC GACAT CTCTG CAGTC GGGTA GTTAA ACCGA CCTTC AGACA TAGGC AGGCG TATAT CTTGT GACGG TAAGC TTGGC AC-3 '(SEQ ID NO: 1) For uranil ions having base sequences and based on recently reported (Liu et al. , PNAS , 2007 , 104 , 2056-2061) in vitro-selected uranil ion-specific catalytic DNA (or simply DNAzyme) Produced by alteration of catalytic sensor

Uranyl ion-specific sensors reported in this document include DNA enzyme strands with 3 'quencher and central ribonucleotide adenosine (riboA) and DNA substrates with fluorophores and quencher at 3' and 5 'ends, respectively. In contrast, the 5'-thiol-containing DNA aptamer of the present invention was replaced with deoxyribonucleotide adenosine riboA in the reported uranyl ion-specific DNAzyme. The uranil ion-specific catalytic sensor of this document is characterized in that when uranil ions bind, they are cleaved at the central ribonucleotide adenosine site, whereas the uranil ion-specific DNA based aptamer of the present invention is directed to uranil ions. Since the purpose is to specifically bind and separate the uranyl ions from the environment, the central ribonucleotide adenosine site is replaced with deoxyribonucleotide adenosine so as not to be cleaved by binding with the uranyl ion.

The uranil ion-specific DNA aptamer of the present invention, HS-DNA 1 was prepared using a primer having a thiol group at the 5 'end and a polymerase chain reaction (PCR). The aptamer solution contained in the dialysis casing (cutoff molecular weight 10,000) was equilibrated against the solution containing metal ions (uranyl acetate (2.1 μM), NaHCO ₃ (21 mM), and HEPES (0.1 M), pH 8.01). Wherein NaHCO ₃ was added to promote solubilization of uranil ions at pH 8.01, such that uranil ions were predominantly present as UO ₂ (CO ₃ ) ₃ ^4- in solution. Commercially available dialysis casings (Slide-A-Lyzer Dialysis Cassettes, Thermo, Bellefonte, PA) were used to minimize concentration changes due to osmotic pressure. After equilibration was taken, the concentration of metal ions outside the dialysis casing was measured by inductively coupled plasma mass spectrometry (ICP-MS) to calculate the amount of metal ions bound to the aptamer HS-DNA 1. The graph of FIG. 1 consists of two straight lines intersecting at [HS-DNA 1 ] ₀ equivalent to [UO ₂ ²⁺ ] ₀ , which shows that the aptamer forms a 1: 1 type complex with uranil ions. Importantly, the inventors conducted HPLC experiments with anion-exchange columns before and after uranyl ion binding, and, unlike the reported UO ₂ ²⁺ -specific sensors, the HS-DNA 1 of the present invention is uranyl ion-bonded. It was found that no magnetic cleavage was caused by. That is, the uranil ion-specific DNA-based aptamers of the present invention bind very strongly and selectively to uranil ions due to their high sensitivity and high selectivity to uranil ions, while not cleaved by the binding of uranil ions. Therefore, it can be usefully used for separation of uranyl ions.

On the other hand, the aptamer was conjugated with the solid support in order to facilitate separation of the uranil ion-aptamer complex after binding to the uranil ion. Aptamer-SMCC-PS, an aptamer conjugated to a solid support, is an aminopolystyrene (aminoPS) resin (polystyrene-co-vinylbenzylamine-co-divinylbenzene, mesh, used as a solid support, according to the schematic of FIG. : 100-200, 1.0 mmol N / g resin, Sigma) was prepared by the modification. The amount of sulfo-SMCC (sulfosuccinimidyl 4- ( N -maleimidomethyl) cyclohexane-1-carboxylate, Sigma) added to aminoPS during the synthesis step was exposed to attack by excess sulfo-SMCC. It was 5 mol% of an amino group. The amount of HS-DNA 1 conjugated with the resin was estimated to be approximately 2.65 nmol / g resin, ie 0.265 mol% of amino groups were covalently attached to HS-DNA 1 .

The amount of uranyl ions that can be bound to the aptamer-SMCC-PS was measured using a fixed amount of aptamer-SMCC-PS (FIG. 4). Approximately 20 mg of modified resin is suspended in 1 mL (pH 8.01) of a solution of uranyl acetate, NaHCO ₃ (21 mM), and HEPES (0.1 M), and the mixture is kept at 25 ° C. at 100 rpm for 2 days. Shaken. The beads collected by filtration were washed three times with buffer solution (0.55 M NaCl, HEPES 0.01 M , pH 8.01; 1 mL) over 3 hours to allow UO ₂ (CO ₃₎ to be bound by the resin via simple adsorption. ) ₃ ^4- was removed. As confirmed by ICP-MS, three treatments with NaCl were sufficient to remove loosely bound uranium species. The beads were washed three more times with distilled water (2 mL) and then they were washed with 1 N HCl aqueous solution (2 mL). The amount of uranyl ions released by HCl treatment was measured by ICP-MS. Analysis of ligand binding experiments can be based on a simple model called the law of mass action. Fractional occupancy or binding coefficient can be defined as the fraction of all receptors bound to uranyl ions as described in Equation 1 below, with an apparent dissociation of about 84.6 fM in the presence of 21 mM bicarbonate ions at pH 8.01. Estimation of the constant K _d ^app is possible (FIG. 4).

Equation 1

However, in the case of insoluble chelating agents of uranyl ions, the formation constant of the uranyl complex (K _f) Is precisely inferred by analogy with the Langmuir isotherm for gas adsorption on solid surfaces.k _adOfk _deIt can be represented by (Formula 2). Uranyl Complex (UO)₂ ²⁺Complexation of uranyl ions by the binding site (BS) to form BS) can be further assumed by analogy using the Langmuir isotherm, independent of subsequent binding.K _fDirect from the equilibrium concentration of uncomplexed uranil ions whenK _fIt is not possible to measure. instead,K _fIs the equilibrium constant for the exchange reaction represented by equation 3, which is a combination ofK _ex=k _OneOfk _-One) Can be estimated indirectly by measuringK _fendK _exAndK _f ^carb(10^21.54Can be calculated from In the following formula, [BS], [BS]₀, And [UO₂ ²⁺BS] is BS and UO₂ ²⁺The concentration of BS obtainable when the BS is assumed to dissolve, the concentration initially added in the BS, and the UO₂ ²⁺Each concentration of BS is shown. The initially added concentration of uranil ions is [UO₂ ²⁺]₀It is expressed as [HCO₃ ^-] >> [UO₂ ²⁺]₀[UO experimentally measured under the conditions of₂ ²⁺From [BS] and [CO used for measurement₃ ^2-], [UO₂ ²⁺]₀, And [BS]₀From the value ofK _exThe value of[HCO₃ ^-] HCO₃ ^-]₀It can be calculated from Equation 5 based on the assumption that can be approximated with. log for Aptamer-SMCC-PS in the presence of 21 mM bicarbonate ions at pH 8.10 and 25 ° C.K _fThe value of was 22.9 ± 1.2.K _d ^appWowK _fThe difference between the estimates of[UO₂ ²⁺]₀It is thought to be due to experimental error caused by the relatively inferior sensitivity of ICP-MS in.

Equation 2

Expression 3

Equation 4

Equation 5

In order to satisfy the economic ease, it is known that more than 500 mg of uranium per gram of resin is required per day. In addition, the chelating agent must be recycled several times. To date, no uranyl chelating agents have been devised that meet these criteria. The results of the present invention indicate that in order to increase the mol% of HS-DNA 1 on the surface of the resin, it is necessary to raise the content of the DNA aptamer in the aptamer-SMCC-PS resin, but the high log K _f value and the stability of the DNA Due to this well shows that the DNA aptamer of the present invention can meet the economic criteria required for the extraction of uranium from seawater and the like.

The present invention also provides a method for separating uranil ions from a sample that is believed to contain uranil ions using an uranil ion-specific DNA based aptamer.

The method of the present invention can utilize any DNA-based aptamer that specifically binds to uranil ions, and in particular, effectively utilizes uranil ions using HS-DNA 1, the uranil ion-specific DNA aptamer described above. Can be separated

The method of the present invention includes contacting a uranil ion-specific DNA aptamer with a sample believed to contain uranyl ions, and separating the uranil ion-DNA aptamer complex.

In the method of the present invention, the uranyl ion-specific DNA aptamer is more preferably in a form conjugated to a solid support in order to facilitate separation of the complex after binding to the uranyl ion.

The uranil ion-specific DNA aptamer of the present invention binds with high specificity to uranil ions and is not cleaved by the binding of uranil ions, thereby not being affected by the interference of various other metal ions in an environment such as seawater. Uranyl ions can be separated with high efficiency, thereby enabling more efficient uranium acquisition at a lower cost than conventional uranium acquisition methods.

1 shows the structure of uranyl ion-specific DNA aptamer HS-DNA 1.

FIG. 2 shows the results of uranil ion binding experiments of HS-DNA 1 showing that HS-DNA 1 forms a 1: 1 type complex with uranil ions.

3 shows a synthetic scheme of aptamer-SMCC-PS resin.

4 shows the apparent dissociation and formation constants of aptamer-SMCC-PS resins for uranyl ions are 84.6 fM and 10 ^{(22.9 ± 1.2)} , respectively, in the presence of 21 mM bicarbonate ions. Uranyl ion bond test results of -PS resin.

Hereinafter, the present invention will be described in more detail through experimental examples. The following experimental examples are intended to illustrate the invention and should not be understood as limiting the invention in any way.

Experimental Example 1. Preparation of HS-DNA 1

Reagents were purchased from the vendor and used without further purification and double distilled water was used for all experiments. Aminopolystyrene was purchased from Invitrogen (Carlsbad, Calif.) And Uranyl Acetate Dihydrate (UO ₂ (CH ₃ COO) ₂ · 2H ₂ O, 99.0%) from Merck was used as a source of uranyl ion without further purification. It was. The crosslinker used was sulfosuccinimidyl 4- ( N -maleimidomethyl) cyclohexane-1-carboxylate (sulfo-SMCC, Sigma). DNA and uranium concentrations were measured by absorbance at 260 nm using an Agilent 8453 UV / Vis spectrophotometer and by ICP-MS using the PerkinElmer ELAN6100 model, respectively.

HS-DNA 1 (5'-HS- (CH ₂ ) ₆ -CTGCA GAATT CTAAT ACGAC TCACT ATAGG AAGAG ATGGC GACAT CTCTG CAGTC GGGTA GTTAA ACCGA CCTTC AGACA TAGGC AGGCG TATAT CTTGT GACGG TAAGC TTGGC AC-3 ') It was synthesized using 117 mer antisense oligonucleotides containing the T7 promoter sequence at the '-HS- (CH ₂ ) ₆ -CTGCA GAATT CTAAT-3') (SEQ ID NO: 2) and at the 5'-end, both of which were integrated DNA Technologies Purchased from

Experimental Example 2 Preparation of DNA-Based Aptamer Conjugated with AminoPS Resin

In order to construct an effective immobilized uranophile using HS-DNA 1 , DNA aptamer was introduced into the solid support to generate aptamer-SMCC-PS as shown in the schematic of FIG. 3. Aptamer-SMCC-PS is an aminopolystyrene (aminoPS) resin (polystyrene-co-vinylbenzylamine-co-divinylbenzene, mesh: 100-200, 1.0 mmol N / used as a solid support according to the schematic of FIG. Gram resin, Sigma). The amount of sulfo-SMCC (sulfosuccinimidyl 4- ( N -maleimidomethyl) cyclohexane-1-carboxylate, Sigma) added to aminoPS during the synthesis step was determined by Zhang et al. , Org. Lett. 2001 . , 3 , 275-278; Derfus et al. , Bioconjug. Chem. 2007 , 18 , 1391-1396) 5 mol% of amino groups exposed to attack by excess sulfo-SMCC. The amount of HS-DNA 1 conjugated with the resin was determined to be approximately 2.65 nmol / g resin, ie 0.265 mol% of amino groups were covalently attached to HS-DNA 1 .

Experimental Example 3. Uranyl Ion Bonding Experiment

The amount of uranyl ions that can be bound to aptamer-SMCC-PS was measured in a fixed amount of aptamer-SMCC-PS (FIG. 4). Approximately 20 mg of modified resin was suspended in 1 ml of uranil acetate solution, NaHCO ₃ (21 mM), and HEPES (0.1 M), pH 8.01. The mixture was shaken at 50 X g , 25 ° C for 2 days. The beads collected by filtration were washed three times with buffer solution (0.55 M NaCl, HEPES 0.01 M , pH 8.01; 1 ml) over 3 hours, to allow UO ₂ (CO ₃ ) to be bound to the resin by simple adsorption. ₃ ^4- was removed. As confirmed by ICP-MS, three treatments with NaCl were sufficient to remove loosely bound uranium species. The beads were washed three more times with distilled water (2 ml), then the beads were washed with 1 N HCl aqueous solution (2 ml). The amount of uranyl ions released by HCl treatment was measured by ICP-MS.

As a result, it was found that about 0.63 micrograms of uranium can be complexed to 1 g of resin, demonstrating that all DNA aptamers introduced into the resin can bind strongly to uranyl ions. The apparent dissociation constant (K _d ^app ) and formation constant (K _f ) for the uranil complex of the resin are about 84.6 fM K _d ^app and about 22.9 ± 1.2 in the presence of 21 mM bicarbonate ions at pH 8.01. LogK _{f of} was obtained. The results of this invention suggest that alteration of the aptamer-containing resin will improve the uranil-binding capacity, thereby enabling economic recovery of uranium from seawater.

Claims

Uranyl ion-specific DNA aptamers.
The uranyl ion-specific DNA aptamer according to claim 1, having the structure of FIG. 1.
The uranyl ion-specific DNA aptamer according to claim 1, having a nucleotide sequence of SEQ ID NO: 1.
The uranyl ion-specific DNA aptamer according to any one of claims 1 to 3, which is conjugated to a solid support.
5. The uranil ion-specific DNA aptamer according to claim 4, wherein the solid support is an aminopolystyrene resin.
6. The uranil ion-specific DNA aptamer according to claim 5, wherein the solid support is sulfosuccinimidyl 4- ( N -maleimidomethyl) cyclohexane-1-carboxylate-aminopolystyrene resin.
Separating the uranil ions from the sample, comprising contacting the uranil ion-specific DNA aptamer with a sample believed to contain uranyl ions, and separating the uranil ion-DNA aptamer complex. Way.
8. The method of claim 7, wherein the uranil ion-specific DNA aptamer has the structure of FIG.
8. The method of claim 7, wherein the uranil ion-specific DNA aptamer has the nucleotide sequence of SEQ ID NO: 1.
10. The method of any one of claims 7 to 9, wherein the uranil ion-specific DNA aptamer is conjugated to a solid support.
The method of claim 10 wherein the solid support is an aminopolystyrene resin.
12. The method of claim 11, wherein the solid support is sulfosuccinimidyl 4- ( N -maleimidomethyl) cyclohexane-1-carboxylate-aminopolystyrene resin.