Bioorganic & Medicinal Chemistry Letters 21 (2011) 4020–4022

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Bioorganic & Medicinal Chemistry Letters

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Binding of uranyl ion by a DNA aptamer attached to a solid support

Jisu Kim, Min Young Kim, Hoon Sik Kim, Sang Soo Hah ⇑Department of Chemistry, Research Institute for Basic Sciences, and Research Center for New Nano Bio Fusion Technology, Kyung Hee University, 1 Hoegi-dong,Dongdaemun-gu, Seoul 130-701, Republic of Korea

a r t i c l e i n f o

Article history:Received 21 January 2011Revised 11 April 2011Accepted 29 April 2011Available online 6 May 2011

Keywords:Uranyl ionUranophileAptamer

0960-894X/$ - see front matter � 2011 Elsevier Ltd.doi:10.1016/j.bmcl.2011.04.139

⇑ Corresponding author. Tel.: +82 2 961 2186; fax:E-mail address: sshah@khu.ac.kr (S.S. Hah).

a b s t r a c t

A UO2þ2 -specific DNA aptamer was attached to aminopolystyrene (aminoPS) using sulfo-SMCC as a cross-

linking agent in view of high affinity of DNA for uranyl ion. Capacity of the aptamer-conjugated aminoPSresins for uranyl uptake was measured, revealing that about 0.63 lg of uranium can be complexed to 1 gof the resins, which clearly demonstrates that most of DNA aptamers introduced to the resins canstrongly bind to uranyl ion. In the presence of 21 mM bicarbonate ion at pH 8.01, apparent dissociationconstant ðKapp

d Þ of about 84.6 pM and log formation constant (Kf) of about 22.9 were obtained. Results ofthe present study strongly suggest that modification of the aptamer-containing resins can improveuranyl-binding ability, probably leading to economical recovery of uranium from seawater.

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Metal sequestering from water is important for environmentalprotection and recovery of resources.1 Especially, metal-specificchelating resins are much more effective than simple ionexchange resins in metal sequestering as they manifest higherselectivity and greater complexation constants toward targetmetal ions and they are useful in practical applications such astreatment of drinking or waste water as well as extraction ofthe target metal ions from seawater.2 From this point of view,the design of effective host molecules for uranyl ion UO2þ

2 is inev-itably connected with the economic importance of selectiveextraction of uranium from seawater, where uranium existsmainly as the tricarbonato complex of uranyl ion UO2ðCO3Þ4�3 .1,3

Examples of well-studied ligands of uranyl ion are carboxylatesincluding EDTA analogues,4 phenols,1c,5 and b-ketones.1b,6 How-ever, these ligands have turned out to unsatisfactorily lack therequired specificity for uranyl ion due to interference with othermetal ions. As a result, efforts have been directed towards selec-tive molecular recognition of uranyl ion with macrocyclic hostmolecules including crown ethers and calixarenes.1,6,7

To design effective immobile uranophiles, we designed a newUO2þ

2 -specific DNA-based aptamers (HS-DNA 1) for specific andstrong uranyl ion binding. Aptamers are known to be a special classof nucleic acids that can specifically bind, with high affinity, to atarget molecule.8 Thus, they have been used in many bioanalyticalapplications, such as for specific detection of proteins,8,9 metalions,10 and small molecules,11 and for target-specific delivery.12

In particular, a catalytic sensor for uranyl ion based on anin vitro-selected UO2þ

2 -specific catalytic DNA (or simply DNAzyme)

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+82 2 966 3701.

was recently reported,13 which prompted us to prepare UO2þ2 -

specific DNA-based aptamers by a simple modification. Whereasthe reported UO2þ

2 -specific sensor consists of a DNA enzyme strandwith a 30 quencher and a DNA substrate with a ribonucleotideadenosine (riboA) in the middle and a fluorophore and a quencherat the 30 and 50 ends, respectively, our 50-thiol-containing DNAaptamer (HS-DNA 1) was prepared by simply replacing riboA inthe reported UO2þ

2 -specific DNAzyme with deoxyribonucleotideadenosine. In this respect, it was expected that our UO2þ

2 -specificDNA-based aptamer could very strongly and selectively bind touranyl ion due to the part-per-trillion sensitivity or detection limit(�45 pM) and million-fold selectivity over other metal ions of thereported UO2þ

2 -specific sensor.13 In the present study, preparationand uranyl-binding features of the polymer-supported HS-DNA 1are reported together with the results of uranium extraction.

HS-DNA 1 was prepared using polymerase chain reaction(PCR) and a primer with the thiol group at the 50 end, and thebinding study of uranyl ion to HS-DNA 1.14 As shown in Figure1, two straight lines resulting from the binding study intersectat [HS-DNA 1]0 equivalent to ½UO2þ

2 �0, which indicates that the

Figure 2. Uranyl ion binding experiment of aptamer-SMCC-PS (square) andcontrol-SMCC-PS (circle) resins. Curve for aptamer-SMCC-PS was obtained byfitting the data to Eq. 1. The data from the binding experiment of aptamer-SMCC-PSresins indicate that apparent dissociation constant ðKapp

d Þ and formation constant(Kf) for uranyl complexes of the modified resins were estimated to be about 84.6 pMand about 1022.9 ± 1.2, respectively, in the presence of 21 mM bicarbonate ion at pH8.01.

Figure 1. Uranyl ion binding experiment of HS-DNA 1, demonstrating that HS-DNA1 forms 1:1-type complexes with uranyl ion. The aptamer solution contained in thedialysis caging (cutoff M.W. 10,000) was equilibrated against a solution containingthe metal ion (uranyl acetate (2.1 lM), NaHCO3 (21 mM), and HEPES (0.1 M) at pH8.01). After equilibrium reached, the amount of the metal ion bound to HS-DNA 1was calculated by the inductively coupled plasma mass spectroscopy (ICP-MS)measurement of the concentration of the metal ion outside the dialysis caging.

J. Kim et al. / Bioorg. Med. Chem. Lett. 21 (2011) 4020–4022 4021

aptamer forms 1:1-type complexes with uranyl ion and that theformation constant (Kf) for the UO2þ

2 complexes of the DNA apt-amer is too large to be measured from the data of Figure 1,although Kf could be estimated as discussed below. Importantly,we carried out HPLC experiments using anion-exchange columnbefore and after 1-day uranyl ion binding, and found no self-cleavage of HS-DNA 1 caused by the uranyl ion binding, differ-ently from the reported UO2þ

2 -specific sensor.13

In order to construct effective immobile uranophiles using HS-DNA 1, the DNA aptamer was introduced to a solid support, leadingto aptamer-SMCC-PS as illustrated in Scheme 1.14 As a non-specificuranyl-binding control, 50-mer oligonucleotide containing a 50-thiol group at the 50-termini was similarly conjugated with theaminoPS resin (control-SMCC-PS resin).14 The amounts of uranylion that could be bound to the resulting resins were measured withthe fixed amounts of control-SMCC-PS and aptamer-SMCC-PS,respectively (Fig. 2), clearly demonstrating that uranyl ion couldbe specifically bound to the DNA aptamer. Analysis of ligand bind-ing experiments may be based on a simple model, called the law ofmass action. Fractional occupancy or binding coefficient can be de-fined as the fraction of all receptors that are bound to uranyl ion asdescribed in Eq. 1, having allowed for estimation of apparent disso-ciation constant ðKapp

d Þ of about 84.6 pM in the presence of 21 mM

Scheme 1. Synthetic scheme for conjugating of aminopolystyrene (aminoPS) resinswith sulfo-SMCC (sulfosuccinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carbox-ylate) and HS-DNA 1.

bicarbonate ion at pH 8.01, since fractional occupancy is 0.5 when[Ligand] = Kapp

d (Fig. 2).

Fractional Occupancy ¼ ½Ligand�½Ligand� þ Kapp

d

ð1Þ

For insoluble sequestering agents of uranyl ion, however, theformation constant (Kf) for the uranyl complex may be preciselyexpressed as kad/kde (Eq. 2) by analogy with Langmuir isothermfor gas adsorption to solid surfaces.9d,15 It may be further assumedthat complexation of uranyl ion by a binding site (BS) to form theuranyl complex ðUO2þ

2 � BSÞ is independent of succeeding bindings,again by analogy with Langmuir isotherm. It is not possible tomeasure Kf directly from the equilibrium concentration of theuncomplexed uranyl ion when Kf is very large. Instead, Kf can beindirectly estimated by measuring the equilibrium constant(Kex = k1/k�1) for the exchange reaction indicated by Eq. 3 whichis combination of equilibrium processes of Eqs. 2 and 4. As sum-marized in Eq. 6, Kf can be calculated from Kex and Kcarb

f (1021.54 inRef. 16). In the equations, [BS], [BS]0, and ½UO2þ

2 � BS� represent theconcentration of BS (i.e., the concentration of HS-DNA 1 in thisstudy),14 the initially added concentration of BS and the concen-tration of UO2þ

2 � BS, respectively, obtainable when BS andUO2þ

2 � BS are assumed to be dissolved. The initially added concen-tration of uranyl ion is expressed as ½UO2þ

2 �0. From ½UO2þ2 � BS�mea-

sured experimentally under the conditions of ½HCO�3 � � ½UO2þ2 �0

and from the values of ½CO2�3 �, ½UO2þ

2 �0, and [BS]0 employed inthe measurements, the values of Kex can be calculated in termsof Eq. 5, based on an assumption that ½HCO�3 � can be approximatedto ½HCO�3 �0. The value of log Kf for aptamer-SMCC-PS in thepresence of 21 mM bicarbonate ion at pH 8.01 and 25 �C has beenfound to be 22.9 ± 1.2.

UO2þ2 þ BS ¢

kad

kde

UO2þ2 � BS Kf ¼ kad=kde ð2Þ

UO2þ2 � BSþ 3CO2�

3 ¢k1

�k1

UO2ðCO3Þ4�3 þ BS ð3Þ

UO2þ2 þ 3CO2�

3 ¢kcarb

fUO2ðCO3Þ4�3 ð4Þ

kex ¼ð½BS�0 � ½UO2þ

2 � BS�Þð½UO2þ2 �0 � ½UO2þ

2 � BS�Þ½UO2þ

2 � BS�½CO2�3 �

3 ð5Þ

4022 J. Kim et al. / Bioorg. Med. Chem. Lett. 21 (2011) 4020–4022

K f ¼ Kcarbf =Kex ð6Þ

It has been estimated that extraction of more than 500 mg ofuranium is needed per gram of resin per day to meet the econom-ical feasibility.17,18 In addition, the sequestering agent must berecycled many times. To date, no uranyl sequestering agents meet-ing these criteria have been designed. Although it is needed to raisethe content of DNA aptamers in the aptamer-SMCC-PS resins, thatis, to increase the mol % of HS-DNA 1 on the surface of the resins,our results strongly suggest that the economic criteria can bemet due to the high log Kf value, the relatively easy synthesisand modification of DNA to be conjugated to solid supports, andthe stability of DNA in water. The uranyl-binding ability of theaptamers may be also improved by modifying the electrostaticenvironment on the surface of the resins through introduction ofextra functional groups. By using the method of data analysisand the information provided by the present study, furtherimprovement of the binding ability and selectivity properties ofthe aptamer-based uranophiles is in progress in this laboratory.

Acknowledgments

This work was supported by Basic Science Program through theNational Research Foundation of Korea (KRF) funded by theMinistry of Education, Science and Technology (MEST) (No. 2010-0015218).

References and notes

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13. Liu, J.; Brown, A. K.; Meng, X.; Cropek, D. M.; Istok, J. D.; Watson, D. B.; Lu, Y.Proc. Natl. Acad. Sci. U.S.A. 2007, 104, 2056.

14. Reagents were obtained from commercial suppliers and were used withoutfurther purification, and double-distilled water was used for all experiments.Aminopolystyrene was obtained from Sigma, and uranyl acetate dihydrate(UO2(CH3COO)2�2H2O, 99.0%) from Merck was used as a uranyl ion source,respectively. Double-distilled deionized water was used throughout theexperiments. Cross-linker used was sulfo-SMCC (sulfosuccinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate, Sigma). DNA and uraniumconcentrations were measured by absorbance at 260 nm using Agilent 8453UV–visible spectrophotometer and by inductively coupled plasma massspectroscopy (ICP-MS) using a PerkinElmer ELAN6100 model, respectively.The DNA-based aptamer containing a 50-thiol group at the 50-termini of DNAmolecules (50-HS-CTGCA GAATT CTAAT ACGAC TCACT ATAGG AAGAG ATGGCGACAT CTCTG CAGTC GGGTA GTTAA ACCGA CCTTC AGACA TAGGC AGGCGTATAT CTTGT GACGG TAAGC TTGGC AC-30) was synthesized using the 15-merprimer (50-HS-CTGCA GAATT CTAAT-30) and the 117-mer antisenseoligonucleotide, both purchased from Integrated DNA Technologies. The PCRproducts were purified by gel electrophoresis followed by ethanolprecipitation.The aptamer solution contained in the dialysis caging (cutoff M.W. 10,000) wasequilibrated against a solution containing the metal ion (uranyl acetate(2.1 lM), NaHCO3 (21 mM), and HEPES (0.1 M) at pH 8.01), where NaHCO3 wasadded to facilitate the solubilization of uranyl ion at pH 8.01, thus makinguranyl ion be mainly present as UO2ðCO3Þ4�3 in the solution. Commerciallyavailable dialysis casings (Slide-A-Lyzer G2 Dialysis Cassettes, Thermo) wereused to minimize concentration changes due to osmotic pressure. By ICP-MSmeasurement of the concentration of the metal ion outside the dialysis cagingafter equilibrium was reached, the amount of the metal ion bound to theaptamer was calculated.In order to conjugate the aptamer with the aminopolystyrene (aminoPS) resins(polystyrene-co-vinylbenzylamine-co-divinylbenzene, mesh: 100–200,1.0 mmol N per gram resin, Sigma), the DNA molecules were treated withdithiothreitol (Sigma) and added to the SMCC-activated aminoPS resinaccording to the literature (Ref. 15c). The amount of the HS-DNA 1conjugated with the resins was estimated to be approximately 2.65 nmol/gresin, that is, 0.265 mol % of amino groups were attached to HS-DNA 1. 50-meroligonucleotide containing a 50-thiol group at the 50-termini (50-HS-CCCCCCCCCC CCCCC CCCCC CCCCC CCCCC CCCCC CCCCC CCCCC CCCCC-30) waspurchased from Integrated DNA Technologies and conjugated with theaminoPS resin as described. The amount of the 50-mer DNA conjugated withthe resins was estimated to be approximately 2.23 nmol/g resin. The resultingcontrol-SMCC-PS was used as a control to show whether any of the uranyl ioncould be bound to non-specific DNA.The amounts of uranyl ion that could be bound to control-SMCC-PS andaptamer-SMCC-PS were measured with the fixed amount of control-SMCC-PSand aptamer-SMCC-PS, respectively. Approximately 20 mg of the resin (i.e.,0.112 nmol 50-mer DNA and 0.133 nmol HS-DNA 1, respectively) wassuspended in a 1 ml solution of uranyl acetate, NaHCO3 (21 mM), and HEPES(0.1 M) at pH 8.01. The mixture was shaken for 2 days at 50�g, 25 �C. Thebeads collected by filtration were washed with a buffer solution (0.55 M NaCl,HEPES 0.01 M, pH 8.01; 1 ml) three times over a period of 3 h to removeUO2ðCO3Þ4�3 that might have been bound by the resin through simpleadsorption. Treatment with NaCl three times was sufficient for removal ofloosely bound uranium species as checked by ICP-MS. After the beads werewashed with distilled water (2 ml) thrice more, they were washed with 1 Naqueous HCl solution (2 ml). The amount of uranyl ion released by HCltreatment was measured by ICP-MS.All experiments were performed in duplicate.

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