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Binding of uranyl ion by a DNA aptamer attached to a solid support
Bioorganic & Medicinal Chemistry Letters 21 (2011) 4020–4022
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters journal homepage: www.elsevier.com/locate/bmcl
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 2011 Revised 11 April 2011 Accepted 29 April 2011 Available online 6 May 2011 Keywords: Uranyl ion Uranophile Aptamer
a b s t r a c t A UO2þ 2 -specific DNA aptamer was attached to aminopolystyrene (aminoPS) using sulfo-SMCC as a crosslinking agent in view of high affinity of DNA for uranyl ion. Capacity of the aptamer-conjugated aminoPS resins for uranyl uptake was measured, revealing that about 0.63 lg of uranium can be complexed to 1 g of the resins, which clearly demonstrates that most of DNA aptamers introduced to the resins can strongly bind to uranyl ion. In the presence of 21 mM bicarbonate ion at pH 8.01, apparent dissociation constant ðK app d Þ of about 84.6 pM and log formation constant (Kf) of about 22.9 were obtained. Results of the present study strongly suggest that modification of the aptamer-containing resins can improve uranyl-binding ability, probably leading to economical recovery of uranium from seawater. Ó 2011 Elsevier Ltd. All rights reserved.
Metal sequestering from water is important for environmental protection and recovery of resources.1 Especially, metal-specific chelating resins are much more effective than simple ion exchange resins in metal sequestering as they manifest higher selectivity and greater complexation constants toward target metal ions and they are useful in practical applications such as treatment of drinking or waste water as well as extraction of the target metal ions from seawater.2 From this point of view, the design of effective host molecules for uranyl ion UO2þ 2 is inevitably connected with the economic importance of selective extraction of uranium from seawater, where uranium exists 1,3 mainly as the tricarbonato complex of uranyl ion UO2 ðCO3 Þ4 3 . Examples of well-studied ligands of uranyl ion are carboxylates including EDTA analogues,4 phenols,1c,5 and b-ketones.1b,6 However, these ligands have turned out to unsatisfactorily lack the required specificity for uranyl ion due to interference with other metal ions. As a result, efforts have been directed towards selective molecular recognition of uranyl ion with macrocyclic host molecules including crown ethers and calixarenes.1,6,7 To design effective immobile uranophiles, we designed a new UO2þ 2 -specific DNA-based aptamers (HS-DNA 1) for specific and strong uranyl ion binding. Aptamers are known to be a special class of nucleic acids that can specifically bind, with high affinity, to a target molecule.8 Thus, they have been used in many bioanalytical applications, such as for specific detection of proteins,8,9 metal ions,10 and small molecules,11 and for target-specific delivery.12 In particular, a catalytic sensor for uranyl ion based on an in vitro-selected UO2þ 2 -specific catalytic DNA (or simply DNAzyme)
⇑ Corresponding author. Tel.: +82 2 961 2186; fax: +82 2 966 3701. E-mail address: [email protected] (S.S. Hah). 0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.bmcl.2011.04.139
was recently reported,13 which prompted us to prepare UO2þ 2 specific DNA-based aptamers by a simple modification. Whereas the reported UO2þ 2 -specific sensor consists of a DNA enzyme strand with a 30 quencher and a DNA substrate with a ribonucleotide adenosine (riboA) in the middle and a fluorophore and a quencher at the 30 and 50 ends, respectively, our 50 -thiol-containing DNA aptamer (HS-DNA 1) was prepared by simply replacing riboA in the reported UO2þ 2 -specific DNAzyme with deoxyribonucleotide adenosine. In this respect, it was expected that our UO2þ 2 -specific DNA-based aptamer could very strongly and selectively bind to uranyl ion due to the part-per-trillion sensitivity or detection limit (45 pM) and million-fold selectivity over other metal ions of the 13 reported UO2þ In the present study, preparation 2 -specific sensor. and uranyl-binding features of the polymer-supported HS-DNA 1 are 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 the binding study of uranyl ion to HS-DNA 1.14 As shown in Figure 1, two straight lines resulting from the binding study intersect at [HS-DNA 1]0 equivalent to ½UO2þ 2 0 , which indicates that the
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J. Kim et al. / Bioorg. Med. Chem. Lett. 21 (2011) 4020–4022
Figure 1. Uranyl ion binding experiment of HS-DNA 1, demonstrating that HS-DNA 1 forms 1:1-type complexes with uranyl ion. The aptamer solution contained in the dialysis caging (cutoff M.W. 10,000) was equilibrated against a solution containing the metal ion (uranyl acetate (2.1 lM), NaHCO3 (21 mM), and HEPES (0.1 M) at pH 8.01). After equilibrium reached, the amount of the metal ion bound to HS-DNA 1 was calculated by the inductively coupled plasma mass spectroscopy (ICP-MS) measurement of the concentration of the metal ion outside the dialysis caging.
aptamer forms 1:1-type complexes with uranyl ion and that the formation constant (Kf) for the UO2þ complexes of the DNA apt2 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 column before and after 1-day uranyl ion binding, and found no selfcleavage of HS-DNA 1 caused by the uranyl ion binding, differ13 ently from the reported UO2þ 2 -specific sensor. In order to construct effective immobile uranophiles using HSDNA 1, the DNA aptamer was introduced to a solid support, leading to aptamer-SMCC-PS as illustrated in Scheme 1.14 As a non-specific uranyl-binding control, 50-mer oligonucleotide containing a 50 thiol group at the 50 -termini was similarly conjugated with the aminoPS resin (control-SMCC-PS resin).14 The amounts of uranyl ion that could be bound to the resulting resins were measured with the fixed amounts of control-SMCC-PS and aptamer-SMCC-PS, respectively (Fig. 2), clearly demonstrating that uranyl ion could be specifically bound to the DNA aptamer. Analysis of ligand binding experiments may 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 that are bound to uranyl ion as described in Eq. 1, having allowed for estimation of apparent dissociation constant ðK app d Þ of about 84.6 pM in the presence of 21 mM
Figure 2. Uranyl ion binding experiment of aptamer-SMCC-PS (square) and control-SMCC-PS (circle) resins. Curve for aptamer-SMCC-PS was obtained by fitting the data to Eq. 1. The data from the binding experiment of aptamer-SMCC-PS resins indicate that apparent dissociation constant ðK app Þ and formation constant d (Kf) for uranyl complexes of the modified resins were estimated to be about 84.6 pM 22.9 ± 1.2 and about 10 , respectively, in the presence of 21 mM bicarbonate ion at pH 8.01.
bicarbonate ion at pH 8.01, since fractional occupancy is 0.5 when [Ligand] = K app (Fig. 2). d
Fractional Occupancy ¼
½Ligand ½Ligand þ K app d
For insoluble sequestering agents of uranyl ion, however, the formation constant (Kf) for the uranyl complex may be precisely expressed as kad/kde (Eq. 2) by analogy with Langmuir isotherm for gas adsorption to solid surfaces.9d,15 It may be further assumed that complexation of uranyl ion by a binding site (BS) to form the uranyl complex ðUO2þ 2 BSÞ is independent of succeeding bindings, again by analogy with Langmuir isotherm. It is not possible to measure Kf directly from the equilibrium concentration of the uncomplexed uranyl ion when Kf is very large. Instead, Kf can be indirectly estimated by measuring the equilibrium constant (Kex = k1/k1) for the exchange reaction indicated by Eq. 3 which is combination of equilibrium processes of Eqs. 2 and 4. As summarized in Eq. 6, Kf can be calculated from Kex and K carb (1021.54 in f 2þ Ref. 16). In the equations, [BS], [BS]0, and ½UO2 BS represent the concentration of BS (i.e., the concentration of HS-DNA 1 in this study),14 the initially added concentration of BS and the concentration of UO2þ 2 BS, respectively, obtainable when BS and UO2þ BS are assumed to be dissolved. The initially added concen2 2þ tration of uranyl ion is expressed as ½UO2þ 2 0 . From ½UO2 BS mea2þ sured experimentally under the conditions of ½HCO 3 ½UO2 0 2 2þ and from the values of ½CO3 , ½UO2 0 , and [BS]0 employed in the measurements, the values of Kex can be calculated in terms of Eq. 5, based on an assumption that ½HCO 3 can be approximated to ½HCO 3 0 . The value of log Kf for aptamer-SMCC-PS in the presence of 21 mM bicarbonate ion at pH 8.01 and 25 °C has been found to be 22.9 ± 1.2. kad
2þ UO2þ K f ¼ kad =kde 2 þ BS ¢ UO2 BS kde
k1
4 2 UO2þ 2 BS þ 3CO3 ¢ UO2 ðCO3 Þ3 þ BS k1
kcarb f
4 2 UO2þ 2 þ 3CO3 ¢ UO2 ðCO3 Þ3
Scheme 1. Synthetic scheme for conjugating of aminopolystyrene (aminoPS) resins with sulfo-SMCC (sulfosuccinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate) and HS-DNA 1.
ð1Þ
kex ¼
2þ 2þ ð½BS0 ½UO2þ 2 BSÞð½UO2 0 ½UO2 BSÞ 2 3 ½UO2þ 2 BS½CO3
ð2Þ
ð3Þ
ð4Þ ð5Þ
4022
K f ¼ K carb =K ex f
J. Kim et al. / Bioorg. Med. Chem. Lett. 21 (2011) 4020–4022
ð6Þ
It has been estimated that extraction of more than 500 mg of uranium is needed per gram of resin per day to meet the economical feasibility.17,18 In addition, the sequestering agent must be recycled many times. To date, no uranyl sequestering agents meeting these criteria have been designed. Although it is needed to raise the content of DNA aptamers in the aptamer-SMCC-PS resins, that is, to increase the mol % of HS-DNA 1 on the surface of the resins, our results strongly suggest that the economic criteria can be met due to the high log Kf value, the relatively easy synthesis and modification of DNA to be conjugated to solid supports, and the stability of DNA in water. The uranyl-binding ability of the aptamers may be also improved by modifying the electrostatic environment on the surface of the resins through introduction of extra functional groups. By using the method of data analysis and the information provided by the present study, further improvement of the binding ability and selectivity properties of the aptamer-based uranophiles is in progress in this laboratory. Acknowledgments This work was supported by Basic Science Program through the National Research Foundation of Korea (KRF) funded by the Ministry of Education, Science and Technology (MEST) (No. 20100015218). References and notes 1. (a) Davies, R. V.; Kennedy, J.; McIlroy, R. W.; Spence, R. Nature 1964, 203, 1110; (b) Tabushi, I.; Kobuke, Y.; Nishiya, T. Nature 1979, 280, 665; (c) Shinkai, S.; Koreishi, H.; Ueda, K.; Arimura, T.; Manabe, O. J. Am. Chem. Soc. 1987, 109, 6371; (d) Tabushi, I.; Kobuke, Y.; Ando, K.; Kishimoto, M.; Ohara, E. J. J. Am. Chem. Soc. 1980, 102, 5947. 2. (a) Schmuckler, G. Talanta 1965, 12, 281; (b) Pohllandt, C.; Fritz, J. S. J. Chromatogr. 1979, 176, 189; (c) Kantipuly, C.; Katragadda, S.; Chow, A.; Gesser, H. D. Talanta 1990, 37, 491. 3. Ikeda, A.; Hennig, C.; Tsushima, S.; Takao, K.; Ikeda, Y.; Scheinost, A. C.; Bernhard, G. Inorg. Chem. 2007, 46, 4212. 4. (a) Carey, G. H.; Martell, A. E. J. Am. Chem. Soc. 1968, 90, 32; (b) da Silva, J. J. R. F.; Simoes, M. F. S. J. Inorg. Nucl. Chem. 1970, 32, 1313. 5. Bartusek, M.; Sommer, I. J. Inorg. Nucl. Chem. 1965, 27, 2397. 6. Alberts, A. H.; Cram, D. J. J. Am. Chem. Soc. 1979, 101, 3545. 7. (a) Fux, P.; Lagrange, J.; Lagrange, P. J. Am. Chem. Soc. 1985, 107, 5927; (b) Brighli, M.; Fux, P.; Lagrange, J.; Lagrange, P. Inorg. Chem. 1985, 24, 80; (c) Lagrange, J.; Metabanzoulou, J. P.; Fux, P.; Lagrange, P. Polyhedron 1989, 8, 2251. 8. (a) Nimjee, S. M.; Rusconi, C. P.; Sullenger, B. A. Annu. Rev. Med. 2005, 56, 555; (b) Pavlov, V.; Xiao, Y.; Shlyahovsky, B.; Willner, I. J. Am. Chem. Soc. 2004, 126, 11768. 9. (a) Ho, H. A.; Leclerc, M. J. Am. Chem. Soc. 2004, 126, 1384; (b) Xiao, Y.; Lubin, A. A.; Heeger, A. J.; Plaxco, K. W. Angew. Chem., Int. Ed. 2005, 44, 5456; (c) Balamurugan, S.; Obubuafo, A.; Soper, S. A.; McCarley, R. L.; Spivak, D. A. Langmuir 2006, 22, 6446; (d) Shin, S.; Kim, I.-H.; Kang, W.; Yang, J. K.; Hah, S. S. Bioorg. Med. Chem. Lett. 2010, 20, 3322. 10. Wang, L.; Liu, X.; Hu, X.; Song, S.; Fan, C. Chem. Commun. 2006, 3780; (a) He, F.; Tang, Y.; Wang, S.; Li, Y.; Zhu, D. J. Am. Chem. Soc. 2005, 127, 12343; (b) Ueyama, H.; Takagi, M.; Takenaka, S. J. Am. Chem. Soc. 2002, 124, 14286. 11. (a) Sankaran, N. B.; Nishizawa, S.; Seino, T.; Yoshimoto, K.; Teramae, N. Angew. Chem., Int. Ed. 2006, 45, 1563; (b) Liu, J.; Lu, Y. Angew. Chem., Int. Ed. 2006, 45, 90.
12. Bagalkot, V.; Farokhzad, O. C.; Langer, R.; Jon, S. Angew. Chem., Int. Ed. 2006, 45, 8149. 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 without further purification, and double-distilled water was used for all experiments. Aminopolystyrene was obtained from Sigma, and uranyl acetate dihydrate (UO2(CH3COO)22H2O, 99.0%) from Merck was used as a uranyl ion source, respectively. Double-distilled deionized water was used throughout the experiments. Cross-linker used was sulfo-SMCC (sulfosuccinimidyl 4-(Nmaleimidomethyl)cyclohexane-1-carboxylate, Sigma). DNA and uranium concentrations were measured by absorbance at 260 nm using Agilent 8453 UV–visible spectrophotometer and by inductively coupled plasma mass spectroscopy (ICP-MS) using a PerkinElmer ELAN6100 model, respectively. The DNA-based aptamer containing a 50 -thiol group at the 50 -termini of DNA molecules (50 -HS-CTGCA GAATT CTAAT ACGAC TCACT ATAGG AAGAG ATGGC GACAT CTCTG CAGTC GGGTA GTTAA ACCGA CCTTC AGACA TAGGC AGGCG TATAT CTTGT GACGG TAAGC TTGGC AC-30 ) was synthesized using the 15-mer primer (50 -HS-CTGCA GAATT CTAAT-30 ) and the 117-mer antisense oligonucleotide, both purchased from Integrated DNA Technologies. The PCR products were purified by gel electrophoresis followed by ethanol precipitation. The aptamer solution contained in the dialysis caging (cutoff M.W. 10,000) was equilibrated 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 was added to facilitate the solubilization of uranyl ion at pH 8.01, thus making uranyl ion be mainly present as UO2 ðCO3 Þ4 in the solution. Commercially 3 available dialysis casings (Slide-A-Lyzer G2 Dialysis Cassettes, Thermo) were used to minimize concentration changes due to osmotic pressure. By ICP-MS measurement of the concentration of the metal ion outside the dialysis caging after equilibrium was reached, the amount of the metal ion bound to the aptamer 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 with dithiothreitol (Sigma) and added to the SMCC-activated aminoPS resin according to the literature (Ref. 15c). The amount of the HS-DNA 1 conjugated with the resins was estimated to be approximately 2.65 nmol/g resin, that is, 0.265 mol % of amino groups were attached to HS-DNA 1. 50-mer oligonucleotide containing a 50 -thiol group at the 50 -termini (50 -HS-CCCCC CCCCC CCCCC CCCCC CCCCC CCCCC CCCCC CCCCC CCCCC CCCCC-30 ) was purchased from Integrated DNA Technologies and conjugated with the aminoPS resin as described. The amount of the 50-mer DNA conjugated with the resins was estimated to be approximately 2.23 nmol/g resin. The resulting control-SMCC-PS was used as a control to show whether any of the uranyl ion could be bound to non-specific DNA. The amounts of uranyl ion that could be bound to control-SMCC-PS and aptamer-SMCC-PS were measured with the fixed amount of control-SMCC-PS and 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) was suspended 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 50g, 25 °C. The beads 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 remove UO2 ðCO3 Þ4 that might have been bound by the resin through simple 3 adsorption. Treatment with NaCl three times was sufficient for removal of loosely bound uranium species as checked by ICP-MS. After the beads were washed with distilled water (2 ml) thrice more, they were washed with 1 N aqueous HCl solution (2 ml). The amount of uranyl ion released by HCl treatment was measured by ICP-MS. All experiments were performed in duplicate. 15. (a) Zhang, B.; Cui, Z.; Sun, L. Org. Lett. 2001, 3, 275; (b) Derfus, A. M.; Chen, A. A.; Min, D.-H.; Ruoslahti, E.; Bhatia, S. N. Bioconjug. Chem. 2007, 18, 1391; (c) Kim, I.-H.; Shin, S.; Jeong, Y.-Y.; Hah, S. S. Tetrahedron Lett. 2010, 51, 3446. 16. Atkins, P. W. Physical Chemistry, fourth ed.; Oxford University Press: Oxford, 1990. pp 885–888; (a) Jang, B. B.; Lee, K.-P.; Min, D.-H.; Suh, J. J. Am. Chem. Soc. 1998, 120, 12008. 17. Cinneide, S. O.; Scanlan, J. P.; Hynes, M. J. J. Inorg. Nucl. Chem. 1975, 37, 1013. 18. Kabay, N.; Egawa, H. Sep. Sci. Technol. 1994, 29, 135. and references therein.
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters journal homepage: www.elsevier.com/locate/bmcl
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 2011 Revised 11 April 2011 Accepted 29 April 2011 Available online 6 May 2011 Keywords: Uranyl ion Uranophile Aptamer
a b s t r a c t A UO2þ 2 -specific DNA aptamer was attached to aminopolystyrene (aminoPS) using sulfo-SMCC as a crosslinking agent in view of high affinity of DNA for uranyl ion. Capacity of the aptamer-conjugated aminoPS resins for uranyl uptake was measured, revealing that about 0.63 lg of uranium can be complexed to 1 g of the resins, which clearly demonstrates that most of DNA aptamers introduced to the resins can strongly bind to uranyl ion. In the presence of 21 mM bicarbonate ion at pH 8.01, apparent dissociation constant ðK app d Þ of about 84.6 pM and log formation constant (Kf) of about 22.9 were obtained. Results of the present study strongly suggest that modification of the aptamer-containing resins can improve uranyl-binding ability, probably leading to economical recovery of uranium from seawater. Ó 2011 Elsevier Ltd. All rights reserved.
Metal sequestering from water is important for environmental protection and recovery of resources.1 Especially, metal-specific chelating resins are much more effective than simple ion exchange resins in metal sequestering as they manifest higher selectivity and greater complexation constants toward target metal ions and they are useful in practical applications such as treatment of drinking or waste water as well as extraction of the target metal ions from seawater.2 From this point of view, the design of effective host molecules for uranyl ion UO2þ 2 is inevitably connected with the economic importance of selective extraction of uranium from seawater, where uranium exists 1,3 mainly as the tricarbonato complex of uranyl ion UO2 ðCO3 Þ4 3 . Examples of well-studied ligands of uranyl ion are carboxylates including EDTA analogues,4 phenols,1c,5 and b-ketones.1b,6 However, these ligands have turned out to unsatisfactorily lack the required specificity for uranyl ion due to interference with other metal ions. As a result, efforts have been directed towards selective molecular recognition of uranyl ion with macrocyclic host molecules including crown ethers and calixarenes.1,6,7 To design effective immobile uranophiles, we designed a new UO2þ 2 -specific DNA-based aptamers (HS-DNA 1) for specific and strong uranyl ion binding. Aptamers are known to be a special class of nucleic acids that can specifically bind, with high affinity, to a target molecule.8 Thus, they have been used in many bioanalytical applications, such as for specific detection of proteins,8,9 metal ions,10 and small molecules,11 and for target-specific delivery.12 In particular, a catalytic sensor for uranyl ion based on an in vitro-selected UO2þ 2 -specific catalytic DNA (or simply DNAzyme)
⇑ Corresponding author. Tel.: +82 2 961 2186; fax: +82 2 966 3701. E-mail address: [email protected] (S.S. Hah). 0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.bmcl.2011.04.139
was recently reported,13 which prompted us to prepare UO2þ 2 specific DNA-based aptamers by a simple modification. Whereas the reported UO2þ 2 -specific sensor consists of a DNA enzyme strand with a 30 quencher and a DNA substrate with a ribonucleotide adenosine (riboA) in the middle and a fluorophore and a quencher at the 30 and 50 ends, respectively, our 50 -thiol-containing DNA aptamer (HS-DNA 1) was prepared by simply replacing riboA in the reported UO2þ 2 -specific DNAzyme with deoxyribonucleotide adenosine. In this respect, it was expected that our UO2þ 2 -specific DNA-based aptamer could very strongly and selectively bind to uranyl ion due to the part-per-trillion sensitivity or detection limit (45 pM) and million-fold selectivity over other metal ions of the 13 reported UO2þ In the present study, preparation 2 -specific sensor. and uranyl-binding features of the polymer-supported HS-DNA 1 are 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 the binding study of uranyl ion to HS-DNA 1.14 As shown in Figure 1, two straight lines resulting from the binding study intersect at [HS-DNA 1]0 equivalent to ½UO2þ 2 0 , which indicates that the
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J. Kim et al. / Bioorg. Med. Chem. Lett. 21 (2011) 4020–4022
Figure 1. Uranyl ion binding experiment of HS-DNA 1, demonstrating that HS-DNA 1 forms 1:1-type complexes with uranyl ion. The aptamer solution contained in the dialysis caging (cutoff M.W. 10,000) was equilibrated against a solution containing the metal ion (uranyl acetate (2.1 lM), NaHCO3 (21 mM), and HEPES (0.1 M) at pH 8.01). After equilibrium reached, the amount of the metal ion bound to HS-DNA 1 was calculated by the inductively coupled plasma mass spectroscopy (ICP-MS) measurement of the concentration of the metal ion outside the dialysis caging.
aptamer forms 1:1-type complexes with uranyl ion and that the formation constant (Kf) for the UO2þ complexes of the DNA apt2 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 column before and after 1-day uranyl ion binding, and found no selfcleavage of HS-DNA 1 caused by the uranyl ion binding, differ13 ently from the reported UO2þ 2 -specific sensor. In order to construct effective immobile uranophiles using HSDNA 1, the DNA aptamer was introduced to a solid support, leading to aptamer-SMCC-PS as illustrated in Scheme 1.14 As a non-specific uranyl-binding control, 50-mer oligonucleotide containing a 50 thiol group at the 50 -termini was similarly conjugated with the aminoPS resin (control-SMCC-PS resin).14 The amounts of uranyl ion that could be bound to the resulting resins were measured with the fixed amounts of control-SMCC-PS and aptamer-SMCC-PS, respectively (Fig. 2), clearly demonstrating that uranyl ion could be specifically bound to the DNA aptamer. Analysis of ligand binding experiments may 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 that are bound to uranyl ion as described in Eq. 1, having allowed for estimation of apparent dissociation constant ðK app d Þ of about 84.6 pM in the presence of 21 mM
Figure 2. Uranyl ion binding experiment of aptamer-SMCC-PS (square) and control-SMCC-PS (circle) resins. Curve for aptamer-SMCC-PS was obtained by fitting the data to Eq. 1. The data from the binding experiment of aptamer-SMCC-PS resins indicate that apparent dissociation constant ðK app Þ and formation constant d (Kf) for uranyl complexes of the modified resins were estimated to be about 84.6 pM 22.9 ± 1.2 and about 10 , respectively, in the presence of 21 mM bicarbonate ion at pH 8.01.
bicarbonate ion at pH 8.01, since fractional occupancy is 0.5 when [Ligand] = K app (Fig. 2). d
Fractional Occupancy ¼
½Ligand ½Ligand þ K app d
For insoluble sequestering agents of uranyl ion, however, the formation constant (Kf) for the uranyl complex may be precisely expressed as kad/kde (Eq. 2) by analogy with Langmuir isotherm for gas adsorption to solid surfaces.9d,15 It may be further assumed that complexation of uranyl ion by a binding site (BS) to form the uranyl complex ðUO2þ 2 BSÞ is independent of succeeding bindings, again by analogy with Langmuir isotherm. It is not possible to measure Kf directly from the equilibrium concentration of the uncomplexed uranyl ion when Kf is very large. Instead, Kf can be indirectly estimated by measuring the equilibrium constant (Kex = k1/k1) for the exchange reaction indicated by Eq. 3 which is combination of equilibrium processes of Eqs. 2 and 4. As summarized in Eq. 6, Kf can be calculated from Kex and K carb (1021.54 in f 2þ Ref. 16). In the equations, [BS], [BS]0, and ½UO2 BS represent the concentration of BS (i.e., the concentration of HS-DNA 1 in this study),14 the initially added concentration of BS and the concentration of UO2þ 2 BS, respectively, obtainable when BS and UO2þ BS are assumed to be dissolved. The initially added concen2 2þ tration of uranyl ion is expressed as ½UO2þ 2 0 . From ½UO2 BS mea2þ sured experimentally under the conditions of ½HCO 3 ½UO2 0 2 2þ and from the values of ½CO3 , ½UO2 0 , and [BS]0 employed in the measurements, the values of Kex can be calculated in terms of Eq. 5, based on an assumption that ½HCO 3 can be approximated to ½HCO 3 0 . The value of log Kf for aptamer-SMCC-PS in the presence of 21 mM bicarbonate ion at pH 8.01 and 25 °C has been found to be 22.9 ± 1.2. kad
2þ UO2þ K f ¼ kad =kde 2 þ BS ¢ UO2 BS kde
k1
4 2 UO2þ 2 BS þ 3CO3 ¢ UO2 ðCO3 Þ3 þ BS k1
kcarb f
4 2 UO2þ 2 þ 3CO3 ¢ UO2 ðCO3 Þ3
Scheme 1. Synthetic scheme for conjugating of aminopolystyrene (aminoPS) resins with sulfo-SMCC (sulfosuccinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate) and HS-DNA 1.
ð1Þ
kex ¼
2þ 2þ ð½BS0 ½UO2þ 2 BSÞð½UO2 0 ½UO2 BSÞ 2 3 ½UO2þ 2 BS½CO3
ð2Þ
ð3Þ
ð4Þ ð5Þ
4022
K f ¼ K carb =K ex f
J. Kim et al. / Bioorg. Med. Chem. Lett. 21 (2011) 4020–4022
ð6Þ
It has been estimated that extraction of more than 500 mg of uranium is needed per gram of resin per day to meet the economical feasibility.17,18 In addition, the sequestering agent must be recycled many times. To date, no uranyl sequestering agents meeting these criteria have been designed. Although it is needed to raise the content of DNA aptamers in the aptamer-SMCC-PS resins, that is, to increase the mol % of HS-DNA 1 on the surface of the resins, our results strongly suggest that the economic criteria can be met due to the high log Kf value, the relatively easy synthesis and modification of DNA to be conjugated to solid supports, and the stability of DNA in water. The uranyl-binding ability of the aptamers may be also improved by modifying the electrostatic environment on the surface of the resins through introduction of extra functional groups. By using the method of data analysis and the information provided by the present study, further improvement of the binding ability and selectivity properties of the aptamer-based uranophiles is in progress in this laboratory. Acknowledgments This work was supported by Basic Science Program through the National Research Foundation of Korea (KRF) funded by the Ministry of Education, Science and Technology (MEST) (No. 20100015218). References and notes 1. (a) Davies, R. V.; Kennedy, J.; McIlroy, R. W.; Spence, R. Nature 1964, 203, 1110; (b) Tabushi, I.; Kobuke, Y.; Nishiya, T. Nature 1979, 280, 665; (c) Shinkai, S.; Koreishi, H.; Ueda, K.; Arimura, T.; Manabe, O. J. Am. Chem. Soc. 1987, 109, 6371; (d) Tabushi, I.; Kobuke, Y.; Ando, K.; Kishimoto, M.; Ohara, E. J. J. Am. Chem. Soc. 1980, 102, 5947. 2. (a) Schmuckler, G. Talanta 1965, 12, 281; (b) Pohllandt, C.; Fritz, J. S. J. Chromatogr. 1979, 176, 189; (c) Kantipuly, C.; Katragadda, S.; Chow, A.; Gesser, H. D. Talanta 1990, 37, 491. 3. Ikeda, A.; Hennig, C.; Tsushima, S.; Takao, K.; Ikeda, Y.; Scheinost, A. C.; Bernhard, G. Inorg. Chem. 2007, 46, 4212. 4. (a) Carey, G. H.; Martell, A. E. J. Am. Chem. Soc. 1968, 90, 32; (b) da Silva, J. J. R. F.; Simoes, M. F. S. J. Inorg. Nucl. Chem. 1970, 32, 1313. 5. Bartusek, M.; Sommer, I. J. Inorg. Nucl. Chem. 1965, 27, 2397. 6. Alberts, A. H.; Cram, D. J. J. Am. Chem. Soc. 1979, 101, 3545. 7. (a) Fux, P.; Lagrange, J.; Lagrange, P. J. Am. Chem. Soc. 1985, 107, 5927; (b) Brighli, M.; Fux, P.; Lagrange, J.; Lagrange, P. Inorg. Chem. 1985, 24, 80; (c) Lagrange, J.; Metabanzoulou, J. P.; Fux, P.; Lagrange, P. Polyhedron 1989, 8, 2251. 8. (a) Nimjee, S. M.; Rusconi, C. P.; Sullenger, B. A. Annu. Rev. Med. 2005, 56, 555; (b) Pavlov, V.; Xiao, Y.; Shlyahovsky, B.; Willner, I. J. Am. Chem. Soc. 2004, 126, 11768. 9. (a) Ho, H. A.; Leclerc, M. J. Am. Chem. Soc. 2004, 126, 1384; (b) Xiao, Y.; Lubin, A. A.; Heeger, A. J.; Plaxco, K. W. Angew. Chem., Int. Ed. 2005, 44, 5456; (c) Balamurugan, S.; Obubuafo, A.; Soper, S. A.; McCarley, R. L.; Spivak, D. A. Langmuir 2006, 22, 6446; (d) Shin, S.; Kim, I.-H.; Kang, W.; Yang, J. K.; Hah, S. S. Bioorg. Med. Chem. Lett. 2010, 20, 3322. 10. Wang, L.; Liu, X.; Hu, X.; Song, S.; Fan, C. Chem. Commun. 2006, 3780; (a) He, F.; Tang, Y.; Wang, S.; Li, Y.; Zhu, D. J. Am. Chem. Soc. 2005, 127, 12343; (b) Ueyama, H.; Takagi, M.; Takenaka, S. J. Am. Chem. Soc. 2002, 124, 14286. 11. (a) Sankaran, N. B.; Nishizawa, S.; Seino, T.; Yoshimoto, K.; Teramae, N. Angew. Chem., Int. Ed. 2006, 45, 1563; (b) Liu, J.; Lu, Y. Angew. Chem., Int. Ed. 2006, 45, 90.
12. Bagalkot, V.; Farokhzad, O. C.; Langer, R.; Jon, S. Angew. Chem., Int. Ed. 2006, 45, 8149. 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 without further purification, and double-distilled water was used for all experiments. Aminopolystyrene was obtained from Sigma, and uranyl acetate dihydrate (UO2(CH3COO)22H2O, 99.0%) from Merck was used as a uranyl ion source, respectively. Double-distilled deionized water was used throughout the experiments. Cross-linker used was sulfo-SMCC (sulfosuccinimidyl 4-(Nmaleimidomethyl)cyclohexane-1-carboxylate, Sigma). DNA and uranium concentrations were measured by absorbance at 260 nm using Agilent 8453 UV–visible spectrophotometer and by inductively coupled plasma mass spectroscopy (ICP-MS) using a PerkinElmer ELAN6100 model, respectively. The DNA-based aptamer containing a 50 -thiol group at the 50 -termini of DNA molecules (50 -HS-CTGCA GAATT CTAAT ACGAC TCACT ATAGG AAGAG ATGGC GACAT CTCTG CAGTC GGGTA GTTAA ACCGA CCTTC AGACA TAGGC AGGCG TATAT CTTGT GACGG TAAGC TTGGC AC-30 ) was synthesized using the 15-mer primer (50 -HS-CTGCA GAATT CTAAT-30 ) and the 117-mer antisense oligonucleotide, both purchased from Integrated DNA Technologies. The PCR products were purified by gel electrophoresis followed by ethanol precipitation. The aptamer solution contained in the dialysis caging (cutoff M.W. 10,000) was equilibrated 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 was added to facilitate the solubilization of uranyl ion at pH 8.01, thus making uranyl ion be mainly present as UO2 ðCO3 Þ4 in the solution. Commercially 3 available dialysis casings (Slide-A-Lyzer G2 Dialysis Cassettes, Thermo) were used to minimize concentration changes due to osmotic pressure. By ICP-MS measurement of the concentration of the metal ion outside the dialysis caging after equilibrium was reached, the amount of the metal ion bound to the aptamer 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 with dithiothreitol (Sigma) and added to the SMCC-activated aminoPS resin according to the literature (Ref. 15c). The amount of the HS-DNA 1 conjugated with the resins was estimated to be approximately 2.65 nmol/g resin, that is, 0.265 mol % of amino groups were attached to HS-DNA 1. 50-mer oligonucleotide containing a 50 -thiol group at the 50 -termini (50 -HS-CCCCC CCCCC CCCCC CCCCC CCCCC CCCCC CCCCC CCCCC CCCCC CCCCC-30 ) was purchased from Integrated DNA Technologies and conjugated with the aminoPS resin as described. The amount of the 50-mer DNA conjugated with the resins was estimated to be approximately 2.23 nmol/g resin. The resulting control-SMCC-PS was used as a control to show whether any of the uranyl ion could be bound to non-specific DNA. The amounts of uranyl ion that could be bound to control-SMCC-PS and aptamer-SMCC-PS were measured with the fixed amount of control-SMCC-PS and 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) was suspended 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 50g, 25 °C. The beads 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 remove UO2 ðCO3 Þ4 that might have been bound by the resin through simple 3 adsorption. Treatment with NaCl three times was sufficient for removal of loosely bound uranium species as checked by ICP-MS. After the beads were washed with distilled water (2 ml) thrice more, they were washed with 1 N aqueous HCl solution (2 ml). The amount of uranyl ion released by HCl treatment was measured by ICP-MS. All experiments were performed in duplicate. 15. (a) Zhang, B.; Cui, Z.; Sun, L. Org. Lett. 2001, 3, 275; (b) Derfus, A. M.; Chen, A. A.; Min, D.-H.; Ruoslahti, E.; Bhatia, S. N. Bioconjug. Chem. 2007, 18, 1391; (c) Kim, I.-H.; Shin, S.; Jeong, Y.-Y.; Hah, S. S. Tetrahedron Lett. 2010, 51, 3446. 16. Atkins, P. W. Physical Chemistry, fourth ed.; Oxford University Press: Oxford, 1990. pp 885–888; (a) Jang, B. B.; Lee, K.-P.; Min, D.-H.; Suh, J. J. Am. Chem. Soc. 1998, 120, 12008. 17. Cinneide, S. O.; Scanlan, J. P.; Hynes, M. J. J. Inorg. Nucl. Chem. 1975, 37, 1013. 18. Kabay, N.; Egawa, H. Sep. Sci. Technol. 1994, 29, 135. and references therein.