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Reversible Ag+-crosslinked DNA hydrogels† Cite this: Chem. Commun., 2014, 50, 4065
Weiwei Guo,‡a Xiu-Juan Qi,‡ab Ron Orbach,a Chun-Hua Lu,a Lina Freage,a Iris Mironi-Harpaz,c Dror Seliktar,c Huang-Hao Yangb and Itamar Willner*a
Received 1st December 2013, Accepted 13th February 2014 DOI: 10.1039/c3cc49140d www.rsc.org/chemcomm
DNA hydrogels, consisting of Y-shaped nucleic acid subunits or of nucleic acid-functionalized acrylamide chains, undergo switchable gel-to-solution transitions. The Ag+-stimulated formation of cytosine– Ag+–cytosine complexes results in the crosslinking of the units to yield the hydrogels, while the cysteamine-induced elimination of the Ag+ ions dissociates the hydrogels into a solution phase.
Substantial research efforts have been directed toward the development and applications of hydrogel materials.1,2 Specifically, the development of stimuli-responsive switchable hydrogels, undergoing gel-to-solid or gel-to-solution transitions, has attracted research activities.3 Different stimuli to trigger switchable hydrogel transitions were used, including pH,4 light,5 temperature,6 and host–guest supramolecular interactions.7 Different applications of hydrogels and stimuli-responsive hydrogels were suggested, including controlled drug delivery and release,8 tissue engineering,9 the use of stimuli-controlled hydrogels as pumps or valves,10 and the use of hydrogels as sensors or actuators.11 One specific class of hydrogels consists of DNA-based hydrogels.12 Two general strategies were implemented to develop DNA-based hydrogels. By one method, nucleic acid units are crosslinked by hybridization to form the hydrogel.13 A second approach involves tethering of nucleic acids to hydrophilic polymers and the hydrogel formation through crosslinking by hybridization of the chains.14 Stimuli-responsive hydrogels were also reported. The catalytic one-cycle dissociation of hydrogels by enzymes15 or DNAzymes16 was demonstrated, and cyclic gel-to-solution transitions using switchable pH17 or G-quadruplex18
crosslinking motifs were reported. Different applications of DNA hydrogels were suggested, including the removal of hazardous ions (e.g., Hg2+),19 sensing,20 inscription of structural information,21 switchable fluorescence properties,22 and catalytic DNAzyme functions.18 Different transition metal ions such as Ag+ or Hg2+ are known to bind specifically to nucleotide bases. Nonetheless, to date there have been no reports on the transition-metal-ion-stimulated switchable formation and dissociation of DNA hydrogels. In the present study, we introduce two methods to prepare Ag+-ionsdependent switchable DNA hydrogels that undergo reversible hydrogel–solution transitions by the crosslinking of the hydrogels with Ag+ ions and their dissociation by the removal of the Ag+-ions, using the cysteamine ligand. The crosslinking of the DNA subunits or the nucleic acid modified polymer chains by means of cytosine– Ag+–cytosine bridges23 leads to the formation of the hydrogels, while the elimination of the Ag+ ions from the bridges leads to the dissociation of the hydrogels. The first method to prepare the Ag+-crosslinked hydrogel is schematically presented in Fig. 1. A Y-shaped DNA subunit consisting of the nucleic acids (1), (2) and (3), and a duplex nucleic acid composed of (4) and (5) are used to self-assemble the hydrogel. Each of the three nucleic acids that comprise the Y-shaped unit includes an identical single-strand toehold sequence I, whereas each of the
a
The Institute of Chemistry, The Center for Nanotechnology, The Hebrew University of Jerusalem, Jerusalem, 91904, Israel. E-mail: [email protected]; Fax: +972-2-6527715; Tel: +972-2-6585272 b The Key Laboratory of Analysis and Detection Technology for Food Safety of the MOE, College of Chemistry and Chemical Engineering, Fuzhou University, Fuzhou 350108, China c Faculty of Biomedical Engineering, Technion-Israel Institute of Technology, Haifa 32000, Israel † Electronic supplementary information (ESI) available: Experimental section, Fig. S1–S4. See DOI: 10.1039/c3cc49140d ‡ These authors contributed equally to this work.
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Fig. 1 Ag+-stimulated assembly of a DNA hydrogel and its reversible dissociation. The gel is formed by the Ag+-induced crossing of Y-shaped DNA subunits through the formation of cytosine–Ag+–cytosine bridges. The dissociation of the gel into a DNA solution is stimulated by the removal of the Ag+ ions using the cysteamine ligand.
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nucleic acids in the duplex structure (4)/(5) includes identical toehold sequences I0 . The toehold domain I0 exhibits complementarity to the sequence I, yet these complementary sequences include two C–C mismatches. The base-pairing complementarity of sequences I/I0 is, however, energetically insufficient to stabilize the formation of the respective duplexes. In the presence of Ag+-ions, the formation of the C–Ag+–C bridges stabilizes cooperatively the formation of base-paired duplexes between the sequences I0 and I. Thus, the addition of Ag+-ions to the mixture of the Y-shaped subunits (1)/(2)/(3) and the duplex structures (4)/(5) leads to the crosslinked structure of the DNA hydrogel. Treatment of the hydrogel with cysteamine results in the elimination of the Ag+-ions by the formation of the stable Ag+–cysteamine complex, a process that leads to the dissociation of the bridging units and to the dissociation of the hydrogel. Re-addition of Ag+ restores the crosslinking bridges, and the hydrogel matrix is reformed. Fig. 2(A) shows the macroscopic images of the DNA solution (left) and the resulting hydrogel formed upon the addition of Ag+-ions (right). For three additional reversible hydrogel-DNA solution transitions see images in Fig. S1 (ESI†). Rheological measurements further supported the switchable hydrogel-to-solution transitions of the system. Fig. 2(B) depicts the time-dependent changes in the storage modulus, G0 , and the loss modulus, G00 , upon the addition of Ag+-ions to the mixture of the Y-shaped subunit (1)/(2)/(3) and the duplex (4)/(5). A time-dependent increase in G0 is observed, and this reaches a saturation value of ca. 19 Pa after ten minutes. On the other hand, the loss modulus exhibits a low value, G00 = 7 Pa, which is constant with time. These features, G0 4 G00 , confirm that the system exists in a hydrogel state. It should be noted that at the concentration of Ag+ ions corresponding to 1.8 mM, the duplex structure (4)/(5) is fully utilized to crosslink the Y-shaped subunits. (Note that the
Fig. 2 (A) Images corresponding to the reversible transitions of the system between the solution phase (left) and the hydrogel state (right). (B) Rheology studies demonstrating the time-dependent changes in the G 0 and G00 upon formation of the Ag+-bridged DNA network consisting of the Y-shaped DNA subunits. (C) Cyclic changes in the G 0 and the G00 upon the cyclic transition of the system between the hydrogel state (added Ag+ ions) and solution state (removal of Ag+ ions by cysteamine). (D) Timedependent changes in G 0 corresponding to systems where the Y-shaped units, 0.3 mM, and 0.45 mM of the duplex DNA were subjected to variable concentrations of Ag+: (a) 0 mM, (b) 0.36 mM, (c) 1.35 mM and (d) 1.8 mM. (For the G00 values of the respective systems see Fig. S2, ESI.†)
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concentration of the (4)/(5) bridges is 0.45 mM, and each duplex includes four C–C mismatches to yield the stable Ag+-bridged structure.) Fig. 2(C) shows the cyclic changes in the G0 /G00 values, upon the reversible treatment of the system with Ag+-ions and cysteamine, respectively. While the G0 value switches between high (in the presence of Ag+) and low values (in the presence of cysteamine), the G00 value is almost unaffected by the two triggers. As expected, the stiffness of the resulting hydrogel is controlled by the concentration of added Ag+-ions, Fig. 2(D). Control experiments showed that the hydrogel formation was, indeed, stimulated by bridging the mixture of the Y-shaped subunits and the duplex units by the C–Ag+–C bridges. Treatment of the mixture of the DNA components with other ions, e.g., Hg2+, Fe3+, Ni2+, did not lead to the formation of the hydrogel. Similarly, the substitution of the C–C mismatch existing in the toehold domains I and I0 with other mismatches, e.g., C–T or T–T, did not lead to the formation of hydrogels upon the addition of Ag+-ions. The SEM image of the freeze-dried Ag+-crosslinked DNA hydrogel is presented in Fig. S3 (ESI†). A highly porous matrix is observed, consistent with the formation of a crosslinked nucleic acid network. The second method to synthesize an Ag+-responsive hydrogel is depicted in Fig. 3. Acrylamide monomer units and the acrydite nucleic acid monomer (6) were polymerized to yield copolymer chains consisting of acrylamide units and nucleic acid-functionalized acrylamide units (acrydite nucleic acid (6)). The ratio of the acrylamide : acrydite nucleic acid units corresponded to 140 : 1 (for the determination of the ratio of the components in the copolymer chains see Fig. S4, ESI†). The nucleic acid units tethered to the acrylamide copolymer chains exhibit self-complementarity of base-pairs and two C–C mismatches. The number of complementary base-pairs in the nucleic acid tethers is, however, insufficient to yield stable duplexes at room temperature, and thus, the self-assembly of the hydrogel is prohibited. The addition of Ag+-ions to the system bridges the C–C mismatched pairs to form C–Ag+–C bridges that act cooperatively with the complementary base-pairs to yield energetically stabilized (6)/(6) duplexes between the nucleic acid tethers, leading to the crosslinking of the polymer chains, and to the formation of the
Fig. 3 Ag+-stimulated formation and dissociation of a hydrogel of an acrylamide–acrydite nucleic acid copolymer. The crosslinking of the chains is achieved by bridging the nucleic acids with cytosine–Ag+–cytosine complexes. The separation is induced by the removal of Ag+ ions using cysteamine.
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Fig. 4 (A) Images corresponding to the cyclic solution–hydrogel-solution transitions of the acrylamide–acrydite nucleic acid system upon crosslinking the chains with Ag+ ions and their separation in the presence of cysteamine. (B) Rheology studies corresponding to the time-dependent changes in G 0 and G00 upon the addition of Ag+-ions to the copolymer solution. (C) Cyclic changes in G 0 and G00 upon the formation of the acrylamide–acrydite nucleic acid hydrogel (addition of Ag+ ions), and upon the dissociation of the hydrogel (upon the cysteamine-induced removal of the Ag+ ions). (D) Time-dependent G 0 changes upon subjecting the acrylamide–acrydite nucleic acid chains to variable concentrations of Ag+: (a) 0 mM, (b) 0.08 mM, (c) 0.16 mM and (d) 0.40 mM. (E) SEM image corresponding to the freeze-dried Ag+ crosslinked acrylamide–acrydite nucleic acid hydrogel.
hydrogel matrix. Treatment of the resulting hydrogel with cysteamine excludes the Ag+-ions from the nucleic acid crosslinking units, resulting in the dissociation of the hydrogel. Thus, by the cyclic treatment of the copolymer chains with Ag+-ions and cysteamine, the reversible formation of the hydrogel and its dissociation may be envisaged. Fig. 4(A) depicts the macroscopic images corresponding to the cyclic Ag+-ion-induced formation of the acrylamide–nucleic acid-bridged hydrogel and the subsequent cysteamine-stimulated dissociation of the hydrogel to a polymer solution. Fig. 4(B) shows the time-dependent changes in G 0 and G00 upon addition of Ag+-ions to the acrylamide–acrylamide nucleic acid copolymer solution. A rapid increase in the G 0 is observed, and it reaches a value of ca. 15 Pa, and the G00 value is unaffected upon addition of the Ag+-ions. By the cyclic addition of Ag+-ions and cysteamine, the storage modulus, G 0 , is reversibly switched between high values (17 2 Pa) and low values (1 0.8 Pa), respectively, while the loss modulus is almost unaffected by the addition of Ag+/cysteamine, Fig. 4(C). These results imply that, indeed, Ag+-ions and cysteamine trigger the reversible, and switchable, hydrogel-to-solution transitions. As expected, the stiffness of the resulting hydrogel is controlled by the concentration of Ag+-ions, Fig. 4(D). It should be noted that the molar concentration of the (6)/(6) duplex bridging units is 0.2 mM, and since each duplex includes two C–C mismatches, the Ag+-stimulated bridging units are saturated at a concentration of 0.4 mM of Ag+. We find, however, that increasing the concentration of Ag+ ions beyond the saturation value is
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accompanied by a further increase in the stiffness of the hydrogel. For example, at a concentration of Ag+ ions corresponding to 0.8 mM, the storage modulus increases up to the value of ca. 30 Pa. This is attributed to the cooperative weak bridging of the acrylamide units by Ag+-ions. The formation of the hydrogel is further characterized by the SEM image, Fig. 4(E), which reveals the generation of a porous network. Control experiments further reveal the important functions of the Ag+-ions in the cooperative stabilization of the duplex nucleic acid bridges that crosslink the polymer chains: (i) By the substitution of the C–C mismatches in the bridging units with C–T mismatches, the Ag+ions fail to stimulate the hydrogel formation. (ii) The substitution of the Ag+-ions with other metal ions, e.g., Hg2+, does not lead to formation of a hydrogel. (iii) The formation of the Ag+-induced hydrogel is affected by the ratio of the acrylamide : acrydite nucleic acid units associated with the copolymer chains. Lowering the content of the nucleic acid tethers reduced the quality of the hydrogel, and it exhibited higher fluidity. It should be noted that for both approaches presented in this study to prepare the hydrogels we were able to cycle the formation/dissociation of the hydrogels in the presence of Ag+ ions/cysteamine for six cycles (for the acrylamide co-polymer chain) and three cycles (for the Y-shaped DNA hydrogel) with no noticeable effect on the quality of the hydrogels. Furthermore, the resulting Ag+–cysteamine complex could be eliminated from the hydrogels while recovering the hydrogel components by applying a Microcon spin filter unit (for the detailed procedure see the ESI†). In conclusion, the present study has introduced two different approaches to prepare reversible, and switchable, DNA-based hydrogels using Ag+-ions and cysteamine as triggering stimuli. One method demonstrated the all-DNA formation of a switchable hydrogel using Y-shaped nucleic acid subunits and duplex nucleic acids as crosslinking bridges. The second approach involved the application of acrylamide copolymer chains that were functionalized with nucleic acid tethers. The Ag+-stimulated bridging of the nucleic acid tethers via C–Ag+–C complexes stimulated the crosslinking of the polymer chains, and the formation of the hydrogel. The comparison between the two methods to generate C–Ag+–C crosslinked hydrogels leads to several important conclusions: (i) The all-DNA hydrogel generated by the Ag+-ions crosslinking the Y-shaped DNA subunits, and the nucleic acid-functionalized acrylamide hydrogel generated by the Ag+-ions crosslinking exhibit similar stiffness (G0 = ca. 19 Pa vs. G0 = ca. 15 Pa). (ii) The concentration of Ag+-ions needed to form the stable all-DNA hydrogel is substantially higher than the concentration of Ag+-ions needed to form the high-quality nucleic acidbridged acrylamide hydrogel. This requires the addition of a higher concentration of cysteamine to separate the all-DNA hydrogel. (iii) The overall base concentration of the nucleic acids that yields the Y-shaped crosslinked hydrogels is 65.1 mM, whereas the overall base concentration of the nucleic acids that yields the same volume of acrylamide-crosslinked hydrogel is 4.8 mM. This comparison indicates that the nucleic acid-crosslinked acrylamide hydrogel is more cost-effective. Nonetheless, as such hydrogels may provide stimuli-responsive drug carrying and release matrices, issues of toxicity should be considered too. In this context, the all-DNA hydrogel may reveal superior properties.
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This research is supported by the MICREAgents project (No. 318671) of the EC Seventh Framework Programme and the Volkswagen Foundation, Germany.
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Notes and references 1 (a) K. Y. Lee and D. J. Mooney, Chem. Rev., 2001, 101, 1869; (b) N. A. Peppas, J. Z. Hilt, A. Khademhosseini and R. Langer, Adv. Mater., 2006, 18, 1345; (c) D. Seliktar, Science, 2012, 336, 1124. ¨ller, Angew. Chem., Int. Ed., 2 J. Yuan, D. Wen, N. Gaponik and A. Eychmu 2013, 52, 976. ¨ring, W. Birnbaum and D. Kuckling, Chem. Soc. Rev., 2013, 3 (a) A. Do 42, 7391; (b) A. B. Imran, T. Seki and Y. Takeoka, Polym. J., 2010, 42, 839. 4 S. R. Haines and R. G. Harrison, Chem. Commun., 2002, 2846. 5 J. J. D. de Jong, L. N. Lucas, R. M. Kellogg, J. H. van Esch and B. L. Feringa, Science, 2004, 304, 278. 6 (a) K. Kuroiwa, T. Shibata, A. Takada, N. Nemoto and N. Kimizuka, J. Am. Chem. Soc., 2004, 126, 2016; (b) B. Jeong, S. W. Kim and Y. Bae, Adv. Drug Delivery Rev., 2002, 54, 37. 7 (a) K. Murata, M. Aoki, T. Nishi, A. Ikeda and S. Shinkai, J. Chem. Soc., Chem. Commun., 1991, 1715; (b) J. B. Beck and S. J. Rowan, J. Am. Chem. Soc., 2003, 125, 13922. 8 (a) M. E. Byrne, K. Park and N. A. Peppas, Adv. Drug Delivery Rev., 2002, 54, 149; (b) J. Z. Hilt and M. E. Byrne, Adv. Drug Delivery Rev., 2004, 56, 1599. 9 (a) J. A. Rowley, G. Madlambayan and D. J. Mooney, Biomaterials, 1999, 20, 45; (b) K. Y. Lee and D. J. Mooney, Chem. Rev., 2001, 101, 1869.
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ChemComm 10 (a) D. J. Beebe, J. S. Moore, J. M. Bauer, Q. Yu, R. H. Liu, C. Devadoss and B. H. Jo, Nature, 2000, 404, 588; (b) T. A. Kapur and M. S. Shoichet, J. Biomed. Mater. Res., Part A, 2004, 68, 235. 11 (a) C. Ruan, K. Zeng, O. K. Varghese and C. A. Grimes, Anal. Chem., 2003, 75, 6494; (b) J. H. Holtz and S. A. Asher, Nature, 1997, 389, 829; (c) L. Zhang and R. W. Seitz, Anal. Bioanal. Chem., 2002, 373, 555. 12 (a) Y. H. Roh, R. C. H. Ruiz, S. Peng, J. B. Lee and D. Luo, Chem. Soc. Rev., 2011, 40, 5730; (b) J. Liu, Soft Matter, 2011, 7, 6757. 13 S. H. Um, J. B. Lee, N. Park, S. Y. Kwon, C. C. Umbach and D. Luo, Nat. Mater., 2006, 5, 797. 14 T. Liedl, H. Dietz, B. Yurke and F. Simmel, Small, 2007, 3, 1688. 15 Y. Xing, E. Cheng, Y. Yang, P. Chen, T. Zhang, Y. Sun, Z. Yang and D. Liu, Adv. Mater., 2011, 23, 1117. 16 H. Lin, Y. Zou, Y. Huang, J. Chen, W. Y. Zhang, Z. Zhuang, G. Jenkins and C. J. Yang, Chem. Commun., 2011, 47, 9312. 17 E. Cheng, Y. Xing, P. Chen, Y. Yang, Y. Sun, D. Zhou, L. Xu, Q. Fan and D. Liu, Angew. Chem., Int. Ed., 2009, 48, 7660. 18 C. H. Lu, X. J. Qi, R. Orbach, H. H. Yang, I. Mironi-Harpaz, D. Seliktar and I. Willner, Nano Lett., 2013, 13, 1298. 19 N. Dave, M. Y. Chan, P. J. Huang, B. D. Smith and J. Liu, J. Am. Chem. Soc., 2010, 132, 12668. 20 K. A. Joseph, N. Dave and J. Liu, ACS Appl. Mater. Interfaces, 2011, 3, 733. 21 J. B. Lee, S. Peng, D. Yang, Y. H. Roh, H. Funabashi, N. Park, E. J. Rice, L. Chen, R. Long, M. Wu and D. Luo, Nat. Nanotechnol., 2012, 7, 816. 22 W. Guo, R. Orbach, I. Mironi-Harpaz, D. Seliktar and I. Willner, Small, 2013, 9, 3748. 23 A. Ono, S. Cao, H. Togashi, M. Tashiro, T. Fujimoto, T. Machinami, S. Oda, Y. Miyake, I. Okamoto and Y. Tanaka, Chem. Commun., 2008, 4825.
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Reversible Ag+-crosslinked DNA hydrogels† Cite this: Chem. Commun., 2014, 50, 4065
Weiwei Guo,‡a Xiu-Juan Qi,‡ab Ron Orbach,a Chun-Hua Lu,a Lina Freage,a Iris Mironi-Harpaz,c Dror Seliktar,c Huang-Hao Yangb and Itamar Willner*a
Received 1st December 2013, Accepted 13th February 2014 DOI: 10.1039/c3cc49140d www.rsc.org/chemcomm
DNA hydrogels, consisting of Y-shaped nucleic acid subunits or of nucleic acid-functionalized acrylamide chains, undergo switchable gel-to-solution transitions. The Ag+-stimulated formation of cytosine– Ag+–cytosine complexes results in the crosslinking of the units to yield the hydrogels, while the cysteamine-induced elimination of the Ag+ ions dissociates the hydrogels into a solution phase.
Substantial research efforts have been directed toward the development and applications of hydrogel materials.1,2 Specifically, the development of stimuli-responsive switchable hydrogels, undergoing gel-to-solid or gel-to-solution transitions, has attracted research activities.3 Different stimuli to trigger switchable hydrogel transitions were used, including pH,4 light,5 temperature,6 and host–guest supramolecular interactions.7 Different applications of hydrogels and stimuli-responsive hydrogels were suggested, including controlled drug delivery and release,8 tissue engineering,9 the use of stimuli-controlled hydrogels as pumps or valves,10 and the use of hydrogels as sensors or actuators.11 One specific class of hydrogels consists of DNA-based hydrogels.12 Two general strategies were implemented to develop DNA-based hydrogels. By one method, nucleic acid units are crosslinked by hybridization to form the hydrogel.13 A second approach involves tethering of nucleic acids to hydrophilic polymers and the hydrogel formation through crosslinking by hybridization of the chains.14 Stimuli-responsive hydrogels were also reported. The catalytic one-cycle dissociation of hydrogels by enzymes15 or DNAzymes16 was demonstrated, and cyclic gel-to-solution transitions using switchable pH17 or G-quadruplex18
crosslinking motifs were reported. Different applications of DNA hydrogels were suggested, including the removal of hazardous ions (e.g., Hg2+),19 sensing,20 inscription of structural information,21 switchable fluorescence properties,22 and catalytic DNAzyme functions.18 Different transition metal ions such as Ag+ or Hg2+ are known to bind specifically to nucleotide bases. Nonetheless, to date there have been no reports on the transition-metal-ion-stimulated switchable formation and dissociation of DNA hydrogels. In the present study, we introduce two methods to prepare Ag+-ionsdependent switchable DNA hydrogels that undergo reversible hydrogel–solution transitions by the crosslinking of the hydrogels with Ag+ ions and their dissociation by the removal of the Ag+-ions, using the cysteamine ligand. The crosslinking of the DNA subunits or the nucleic acid modified polymer chains by means of cytosine– Ag+–cytosine bridges23 leads to the formation of the hydrogels, while the elimination of the Ag+ ions from the bridges leads to the dissociation of the hydrogels. The first method to prepare the Ag+-crosslinked hydrogel is schematically presented in Fig. 1. A Y-shaped DNA subunit consisting of the nucleic acids (1), (2) and (3), and a duplex nucleic acid composed of (4) and (5) are used to self-assemble the hydrogel. Each of the three nucleic acids that comprise the Y-shaped unit includes an identical single-strand toehold sequence I, whereas each of the
a
The Institute of Chemistry, The Center for Nanotechnology, The Hebrew University of Jerusalem, Jerusalem, 91904, Israel. E-mail: [email protected]; Fax: +972-2-6527715; Tel: +972-2-6585272 b The Key Laboratory of Analysis and Detection Technology for Food Safety of the MOE, College of Chemistry and Chemical Engineering, Fuzhou University, Fuzhou 350108, China c Faculty of Biomedical Engineering, Technion-Israel Institute of Technology, Haifa 32000, Israel † Electronic supplementary information (ESI) available: Experimental section, Fig. S1–S4. See DOI: 10.1039/c3cc49140d ‡ These authors contributed equally to this work.
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Fig. 1 Ag+-stimulated assembly of a DNA hydrogel and its reversible dissociation. The gel is formed by the Ag+-induced crossing of Y-shaped DNA subunits through the formation of cytosine–Ag+–cytosine bridges. The dissociation of the gel into a DNA solution is stimulated by the removal of the Ag+ ions using the cysteamine ligand.
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nucleic acids in the duplex structure (4)/(5) includes identical toehold sequences I0 . The toehold domain I0 exhibits complementarity to the sequence I, yet these complementary sequences include two C–C mismatches. The base-pairing complementarity of sequences I/I0 is, however, energetically insufficient to stabilize the formation of the respective duplexes. In the presence of Ag+-ions, the formation of the C–Ag+–C bridges stabilizes cooperatively the formation of base-paired duplexes between the sequences I0 and I. Thus, the addition of Ag+-ions to the mixture of the Y-shaped subunits (1)/(2)/(3) and the duplex structures (4)/(5) leads to the crosslinked structure of the DNA hydrogel. Treatment of the hydrogel with cysteamine results in the elimination of the Ag+-ions by the formation of the stable Ag+–cysteamine complex, a process that leads to the dissociation of the bridging units and to the dissociation of the hydrogel. Re-addition of Ag+ restores the crosslinking bridges, and the hydrogel matrix is reformed. Fig. 2(A) shows the macroscopic images of the DNA solution (left) and the resulting hydrogel formed upon the addition of Ag+-ions (right). For three additional reversible hydrogel-DNA solution transitions see images in Fig. S1 (ESI†). Rheological measurements further supported the switchable hydrogel-to-solution transitions of the system. Fig. 2(B) depicts the time-dependent changes in the storage modulus, G0 , and the loss modulus, G00 , upon the addition of Ag+-ions to the mixture of the Y-shaped subunit (1)/(2)/(3) and the duplex (4)/(5). A time-dependent increase in G0 is observed, and this reaches a saturation value of ca. 19 Pa after ten minutes. On the other hand, the loss modulus exhibits a low value, G00 = 7 Pa, which is constant with time. These features, G0 4 G00 , confirm that the system exists in a hydrogel state. It should be noted that at the concentration of Ag+ ions corresponding to 1.8 mM, the duplex structure (4)/(5) is fully utilized to crosslink the Y-shaped subunits. (Note that the
Fig. 2 (A) Images corresponding to the reversible transitions of the system between the solution phase (left) and the hydrogel state (right). (B) Rheology studies demonstrating the time-dependent changes in the G 0 and G00 upon formation of the Ag+-bridged DNA network consisting of the Y-shaped DNA subunits. (C) Cyclic changes in the G 0 and the G00 upon the cyclic transition of the system between the hydrogel state (added Ag+ ions) and solution state (removal of Ag+ ions by cysteamine). (D) Timedependent changes in G 0 corresponding to systems where the Y-shaped units, 0.3 mM, and 0.45 mM of the duplex DNA were subjected to variable concentrations of Ag+: (a) 0 mM, (b) 0.36 mM, (c) 1.35 mM and (d) 1.8 mM. (For the G00 values of the respective systems see Fig. S2, ESI.†)
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concentration of the (4)/(5) bridges is 0.45 mM, and each duplex includes four C–C mismatches to yield the stable Ag+-bridged structure.) Fig. 2(C) shows the cyclic changes in the G0 /G00 values, upon the reversible treatment of the system with Ag+-ions and cysteamine, respectively. While the G0 value switches between high (in the presence of Ag+) and low values (in the presence of cysteamine), the G00 value is almost unaffected by the two triggers. As expected, the stiffness of the resulting hydrogel is controlled by the concentration of added Ag+-ions, Fig. 2(D). Control experiments showed that the hydrogel formation was, indeed, stimulated by bridging the mixture of the Y-shaped subunits and the duplex units by the C–Ag+–C bridges. Treatment of the mixture of the DNA components with other ions, e.g., Hg2+, Fe3+, Ni2+, did not lead to the formation of the hydrogel. Similarly, the substitution of the C–C mismatch existing in the toehold domains I and I0 with other mismatches, e.g., C–T or T–T, did not lead to the formation of hydrogels upon the addition of Ag+-ions. The SEM image of the freeze-dried Ag+-crosslinked DNA hydrogel is presented in Fig. S3 (ESI†). A highly porous matrix is observed, consistent with the formation of a crosslinked nucleic acid network. The second method to synthesize an Ag+-responsive hydrogel is depicted in Fig. 3. Acrylamide monomer units and the acrydite nucleic acid monomer (6) were polymerized to yield copolymer chains consisting of acrylamide units and nucleic acid-functionalized acrylamide units (acrydite nucleic acid (6)). The ratio of the acrylamide : acrydite nucleic acid units corresponded to 140 : 1 (for the determination of the ratio of the components in the copolymer chains see Fig. S4, ESI†). The nucleic acid units tethered to the acrylamide copolymer chains exhibit self-complementarity of base-pairs and two C–C mismatches. The number of complementary base-pairs in the nucleic acid tethers is, however, insufficient to yield stable duplexes at room temperature, and thus, the self-assembly of the hydrogel is prohibited. The addition of Ag+-ions to the system bridges the C–C mismatched pairs to form C–Ag+–C bridges that act cooperatively with the complementary base-pairs to yield energetically stabilized (6)/(6) duplexes between the nucleic acid tethers, leading to the crosslinking of the polymer chains, and to the formation of the
Fig. 3 Ag+-stimulated formation and dissociation of a hydrogel of an acrylamide–acrydite nucleic acid copolymer. The crosslinking of the chains is achieved by bridging the nucleic acids with cytosine–Ag+–cytosine complexes. The separation is induced by the removal of Ag+ ions using cysteamine.
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Fig. 4 (A) Images corresponding to the cyclic solution–hydrogel-solution transitions of the acrylamide–acrydite nucleic acid system upon crosslinking the chains with Ag+ ions and their separation in the presence of cysteamine. (B) Rheology studies corresponding to the time-dependent changes in G 0 and G00 upon the addition of Ag+-ions to the copolymer solution. (C) Cyclic changes in G 0 and G00 upon the formation of the acrylamide–acrydite nucleic acid hydrogel (addition of Ag+ ions), and upon the dissociation of the hydrogel (upon the cysteamine-induced removal of the Ag+ ions). (D) Time-dependent G 0 changes upon subjecting the acrylamide–acrydite nucleic acid chains to variable concentrations of Ag+: (a) 0 mM, (b) 0.08 mM, (c) 0.16 mM and (d) 0.40 mM. (E) SEM image corresponding to the freeze-dried Ag+ crosslinked acrylamide–acrydite nucleic acid hydrogel.
hydrogel matrix. Treatment of the resulting hydrogel with cysteamine excludes the Ag+-ions from the nucleic acid crosslinking units, resulting in the dissociation of the hydrogel. Thus, by the cyclic treatment of the copolymer chains with Ag+-ions and cysteamine, the reversible formation of the hydrogel and its dissociation may be envisaged. Fig. 4(A) depicts the macroscopic images corresponding to the cyclic Ag+-ion-induced formation of the acrylamide–nucleic acid-bridged hydrogel and the subsequent cysteamine-stimulated dissociation of the hydrogel to a polymer solution. Fig. 4(B) shows the time-dependent changes in G 0 and G00 upon addition of Ag+-ions to the acrylamide–acrylamide nucleic acid copolymer solution. A rapid increase in the G 0 is observed, and it reaches a value of ca. 15 Pa, and the G00 value is unaffected upon addition of the Ag+-ions. By the cyclic addition of Ag+-ions and cysteamine, the storage modulus, G 0 , is reversibly switched between high values (17 2 Pa) and low values (1 0.8 Pa), respectively, while the loss modulus is almost unaffected by the addition of Ag+/cysteamine, Fig. 4(C). These results imply that, indeed, Ag+-ions and cysteamine trigger the reversible, and switchable, hydrogel-to-solution transitions. As expected, the stiffness of the resulting hydrogel is controlled by the concentration of Ag+-ions, Fig. 4(D). It should be noted that the molar concentration of the (6)/(6) duplex bridging units is 0.2 mM, and since each duplex includes two C–C mismatches, the Ag+-stimulated bridging units are saturated at a concentration of 0.4 mM of Ag+. We find, however, that increasing the concentration of Ag+ ions beyond the saturation value is
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accompanied by a further increase in the stiffness of the hydrogel. For example, at a concentration of Ag+ ions corresponding to 0.8 mM, the storage modulus increases up to the value of ca. 30 Pa. This is attributed to the cooperative weak bridging of the acrylamide units by Ag+-ions. The formation of the hydrogel is further characterized by the SEM image, Fig. 4(E), which reveals the generation of a porous network. Control experiments further reveal the important functions of the Ag+-ions in the cooperative stabilization of the duplex nucleic acid bridges that crosslink the polymer chains: (i) By the substitution of the C–C mismatches in the bridging units with C–T mismatches, the Ag+ions fail to stimulate the hydrogel formation. (ii) The substitution of the Ag+-ions with other metal ions, e.g., Hg2+, does not lead to formation of a hydrogel. (iii) The formation of the Ag+-induced hydrogel is affected by the ratio of the acrylamide : acrydite nucleic acid units associated with the copolymer chains. Lowering the content of the nucleic acid tethers reduced the quality of the hydrogel, and it exhibited higher fluidity. It should be noted that for both approaches presented in this study to prepare the hydrogels we were able to cycle the formation/dissociation of the hydrogels in the presence of Ag+ ions/cysteamine for six cycles (for the acrylamide co-polymer chain) and three cycles (for the Y-shaped DNA hydrogel) with no noticeable effect on the quality of the hydrogels. Furthermore, the resulting Ag+–cysteamine complex could be eliminated from the hydrogels while recovering the hydrogel components by applying a Microcon spin filter unit (for the detailed procedure see the ESI†). In conclusion, the present study has introduced two different approaches to prepare reversible, and switchable, DNA-based hydrogels using Ag+-ions and cysteamine as triggering stimuli. One method demonstrated the all-DNA formation of a switchable hydrogel using Y-shaped nucleic acid subunits and duplex nucleic acids as crosslinking bridges. The second approach involved the application of acrylamide copolymer chains that were functionalized with nucleic acid tethers. The Ag+-stimulated bridging of the nucleic acid tethers via C–Ag+–C complexes stimulated the crosslinking of the polymer chains, and the formation of the hydrogel. The comparison between the two methods to generate C–Ag+–C crosslinked hydrogels leads to several important conclusions: (i) The all-DNA hydrogel generated by the Ag+-ions crosslinking the Y-shaped DNA subunits, and the nucleic acid-functionalized acrylamide hydrogel generated by the Ag+-ions crosslinking exhibit similar stiffness (G0 = ca. 19 Pa vs. G0 = ca. 15 Pa). (ii) The concentration of Ag+-ions needed to form the stable all-DNA hydrogel is substantially higher than the concentration of Ag+-ions needed to form the high-quality nucleic acidbridged acrylamide hydrogel. This requires the addition of a higher concentration of cysteamine to separate the all-DNA hydrogel. (iii) The overall base concentration of the nucleic acids that yields the Y-shaped crosslinked hydrogels is 65.1 mM, whereas the overall base concentration of the nucleic acids that yields the same volume of acrylamide-crosslinked hydrogel is 4.8 mM. This comparison indicates that the nucleic acid-crosslinked acrylamide hydrogel is more cost-effective. Nonetheless, as such hydrogels may provide stimuli-responsive drug carrying and release matrices, issues of toxicity should be considered too. In this context, the all-DNA hydrogel may reveal superior properties.
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This research is supported by the MICREAgents project (No. 318671) of the EC Seventh Framework Programme and the Volkswagen Foundation, Germany.
Published on 13 February 2014. Downloaded by University of California - Irvine on 27/10/2014 11:47:27.
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