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Cite this: Chem. Commun., 2013, 49, 11308 Received 4th September 2013, Accepted 9th October 2013
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A molecular logical switching beacon controlled by thiolated DNA signals† Cheng Zhang,*a Liuqing Wu,a Jing Yang,b Shi Liub and Jin Xu*a
DOI: 10.1039/c3cc46743k www.rsc.org/chemcomm
A logical switching MB is established, with an ‘‘ON/OFF’’ switching function. In this study, thiolated DNA can participate as a switching controller to regulate the fluorescent increments of other DNA input signals. Assisted by gold nanoparticles and DNA branch migration, one and two-switch systems have been achieved.
As a nanoscale building material, DNA attracts a lot of attention in various applications including assembly materials,1 molecular machines2 and nano-computing.3 Particularly, DNA computing is an emerging research focusing on implementing both logic control and molecular detection. Lots of applications require various advanced methods to monitor or detect molecular outputs by DNA computing systems. One important class of logic computing systems is composed of gold nanoparticles (AuNPs) functionalized with DNA, taking advantage of the abilities of fluorescence quenching and aggregation.4 In fact, AuNP-based molecular beacons (MBs) possess several advantages, such as strong quenching effects, multi-functional methods and sensitive sequence specificity.5 Consequently, different approaches to construct AuNP-based MB are investigated, in which a method of attaching thiolated DNA onto the gold surface is widely used.6 For example, a multicolor nanoscale MB was constructed by Fan and co-workers, using three kinds of thiolated single strand DNA (ssDNA).5a In 2010, Mirkin’s group reported a MB to increase the rate of target hybridization based on an attachment of thiolated ssDNA.7 In previous studies about MBs, the thiol-group modified DNA is used only as a building material to bind DNA onto gold nanoparticles. In addition, triggering input signals for MBs have been limited to nucleic acids, proteins and a few small molecules.8 In order to make the thiol-group as a recognizable signal, it is necessary to establish a relationship between the attachment of the thiol-group and specific output signals. a
Institute of Software, School of Electronics Engineering and Computer Science, Peking University, Key laboratory of High Confidence Software Technologies, Ministry of Education, Beijing, China. E-mail: [email protected], [email protected] b School of Control and Computer Engineering, North China Electric Power University, Beijing, China. Fax: +86-10-61771050; Tel: +86-10-61771050 † Electronic supplementary information (ESI) available: Materials, methods, and experimental details. See DOI: 10.1039/c3cc46743k
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Fig. 1 Principles of switching MB a-B and a-Bs in (a) and (b). A logic diagram is illustrated in (c). Switching results are shown by comparing the values of Q1 and Q2 in (d).
Therefore, it is interesting to explore the possibility of using the thiol-groups as triggers to achieve molecular switching functions. In this study, we prepared a logical switching MB triggered by thiolated DNA inputs. In the MB system, corresponding fluorescent increments can be influenced by an ‘‘ON/OFF’’ switching function, where DNA hairpins B and Bs play the role of the respective triggering signals (Fig. 1). In the ‘‘ON’’ state, treated with thiol-free hairpin B, all DNA inputs can specifically cause an increase of fluorescent signals, without any hindrance. While, in the ‘‘OFF’’ state, treated with thiolated hairpin Bs, the triggering fluorescent increments can be greatly inhibited. In this system, thiolated DNA can participate as a switching controller to regulate the fluorescent increments of DNA inputs. In the first switching MB, the a mode, triggers hairpin B or Bs can hybridize with the thiolated strand A, and the DNA assembly structures can attach onto the surface of AuNPs. Then, assembly structures on MB a-B (‘‘ON’’ state) keep a ‘‘standing’’ state (Fig. 1a), while those on MB a-Bs (‘‘OFF’’ state) slightly change their gestures into a ‘‘lying’’ state (Fig. 1b). It should be noted that hairpin Bs attaches onto AuNPs not only by hybridizing with DNA strand A, but also by a connection of a thiol-group. The hybridization brings the fluorophore (labelled on DNA C-F) into the proximity of the AuNP surface, leading to a significant quenching effect. This journal is
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For the ‘‘ON’’ state, two DNA input signals D and C-z were added into a solution of MB a-B. In the presence of signal D, the assembly structure of hairpin B and C-F can be potentially displaced from AuNPs, leading to an increase of fluorescent signals. In addition, adding input signal C-z can totally displace fluorescent DNA C-F for a longer complementary hybridizing region, thereby also causing an increment of fluorescent signals. In comparison to the ‘‘ON’’ state, for the ‘‘OFF’’ state, adding input signal C-z also leads to an increase in fluorescence. When it comes to addition of signal D, although the displacement happened with a separation between hairpin Bs and strand A, the fluorophore was still in close proximity to AuNPs, because of another connection of the thiol-group from hairpin Bs. Thus, in the case of the ‘‘OFF’’ state, a significant inhibiting effect on the increase in fluorescence should be produced when signal D is added. In other words, in the logic MB a, a switching function for signal D can be achieved by adding thiol-free DNA (‘‘ON’’) or thiolated DNA (‘‘OFF’’). (NOTE: 1) The fluorescence increments were normalized as I = DF/F0, where F0 is the fluorescent intensity in the initial state without adding input strands; DF is the increment of the fluorescence, and average values are obtained by repeating the experiments three times (see ESI†). (2) Taking Imax as a reference, obtained by adding input C-z for a direct and complete displacement, the switching inhibiting effect can be judged by comparing the relative value Q = Iinput/Imax, where Iinput was obtained by adding input D or G. As demonstrated in Fig. 1a, in the ‘‘ON’’ state, in the presence of signals D and C-z, almost the same fluorescence enhancements were observed as I1 = 97.6% and I2 = 95%, respectively (Q1 = 1.02 : 1). However, in the ‘‘OFF’’ state, the fluorescent increment from signal D (I3 = 84.3%) was much less than that of the complete displacement from C-z (I4 = 163.5%) (Q2 = 0.52 : 1). Compared with values of Q1, a great decrease of Q2 may suggest an existence of inhibiting effects in the ‘‘OFF’’ state. To further test whether the switching system can be scaled up to a two-switch system, the second switching MB, the b mode was employed, where an additional displacing site, capable of recognizing the input strand G, was introduced (Fig. 2). In the second switching MB b mode, the DNA assembly structure consists of three strands, named AD, AC and hairpin B/Bs. After hybridizing with fluorescent
DNA C-F, the MB can be triggered by three input signals: D, G and C-z. For the ‘‘ON’’ state (with hairpin B), in the presence of either input signal D or G, the fluorescent DNA complex will dissociate from the AuNPs, thus causing increments of fluorescent signals. On the other hand, treating the MB system with hairpin Bs yields the ‘‘OFF’’ state, where a connection between a thiol-group (hairpin Bs) and an AuNP can be generated. In this case, no matter whether input D or G is added, the fluorescent DNA complex will still attach onto the surface of the AuNPs. Therefore, a significant inhibiting effect on fluorescent enhancement should be produced. In Fig. 2, it can be observed that in the ‘‘ON’’ state, addition of three inputs D, G and C-z has approximately increasing efficiencies of 58.8%, 53% and 55.9%, respectively (Q3 = 1.05 : 0.95 : 1). Almost no difference could be observed among addition of D, G and C-z. While, in the ‘‘OFF’’ state, the fluorescent increments upon adding inputs D and G are 39.2% and 39.2%, respectively, which are much lower than 104.1% upon adding input C-z (Q4 = 0.38 : 0.38 : 1). Compared with Q3 and Q4, the results suggest that a significant inhibiting effect can be induced by the participation of the thiolated DNA signals. It should be noted that, despite having the inhibiting effect in the ‘‘OFF’’ state, the fluorescent increments still existed when adding input strands, suggesting that not all connections were generated between thiolated hairpin Bs and AuNPs. To explore more details of the inhibiting effects induced by thiol-group attachment, it is important to consider efficiencies of such attachment, influenced by structural changes. Fig. 3 shows a schematic diagram of two different ways of attachment with a ‘‘blunt’’ end and a ‘‘protruding’’ end. Here, two kinds of hairpin DNA Bs and B1s are utilized to form two systems: MB b-Bs and MB b-B1s, respectively. In the case of the ‘‘protruding’’ end, the thiolgroup is connected with a flexible ssDNA arm, which is a favorable structure for the attachment. While in the case of the ‘‘blunt’’ end, although the thiol-group is exposed to interact with the surface of the AuNPs, the spatial hindrance of the blunt end of the ssDNA may still impede the attachment of the thiol-group. Using the flexible ‘‘protruding’’ arm, in the ‘‘OFF’’ state, adding inputs D, G and C-z leads to the corresponding fluorescent increments of 57.7%, 56.1% and 60.03% (Q5 = 0.96 : 0.93 : 1). In the ‘‘ON’’
Fig. 2 Principles of switching MB b-B and b-Bs in (a) and (b). A logic diagram is illustrated in (c). Switching results are shown by comparing the values of Q3 and Q4 in (d).
Fig. 3 A schematic diagram of two different ways of attachment with the ‘‘blunt’’ end and the ‘‘protruding’’ end in (a) and (b). Switching results are shown by comparing the values of Q5 and Q6 in (c).
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Fig. 4 Comparisons of the MB b structures of mono-functionalized conjugates (a). Electrophoretic results of 5 nm AuNPs are displayed in 3% agarose gel. (b) MB a-B was purified, and then displaced by adding trigger strands: D (Lane 3) and G (Lane 4). (c) An assembly of a discrete AuNP cluster (5 + 15 nm nanoparticles), and its TEM images.
state of the ‘‘protruding’’ arm, the experimental data were 20.37%, 20.23% and 203.13% (Q6 = 0.1 : 0.1 : 1). Comparing Q4 = 0.38 : 0.38 : 1 and Q6 = 0.1 : 0.1 : 1, it is easy to see that using the ‘‘protruding’’ end has significantly greater inhibiting effects than using the ‘‘blunt’’ end. This phenomenon may be due to that, although the ‘‘blunt’’ end thiol-groups were in the proximity of AuNPs, it is rather difficult for all of them to attach onto the surface of AuNPs because of the strong spatial hindrance. Therefore, the displacements induced by adding inputs D and G will lead to some separation between the fluorophore and AuNPs, and some fluorescent increments will be produced. However, for the ‘‘protruding’’ end, almost all of the thiol-groups were favored to attach onto AuNPs because of their flexible arms, leading to a significant inhibiting effect. Thus, adding inputs D and G will cause a much less fluorescent increment, when compared with using the ‘‘blunt’’ end. In this work, the triggering process of adding input DNA can be confirmed by comparing the MB b structures of mono-functionalized conjugates. By adding different DNA assembly structures: AD, AD + AC, AD + AC + B1 and AD + AC + B1s, the corresponding conjugates with various gel running speeds were generated as shown in Fig. 4a. As expected, the bands of conjugating products shifted slowly as the complexities of DNA assembly structures were increased. Interestingly, we investigated mono-functionalized conjugate MB b-B1 using DNA branch migration, and results of the displacing products in the gel are illustrated (Fig. 4b). As a comparison, MB a-B was purified and displaced by adding strands: D (Lane 3) and G (Lane 4). The increasing gel running speeds of the displacing products suggested that the DNA structures left the AuNPs. In addition, to test whether the bulged DNA loop of hairpin B1 can provide a binding site for further detection, a method of assembly of discrete AuNP clusters was employed. The addition of 5 nm multivalent conjugate B1NP led to a cooperative hybridization with hairpin B1, resulting in the formation of a dimer of gold nanoparticles with asymmetric diameters, 5 and 15 nm (Fig. 4c). In these experiments, we demonstrated the mono-functionalized MB b to be a well-formed conjugate, containing a unique hybridizing site and two triggering toehold regions for strand displacement. In this communication, we present a logical MB system controlled by thiolated DNA input signals, which allows achieving molecular switching functions. Although the ‘‘OFF’’ state was not 11310
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Communication able to completely inhibit enhancement of fluorescent signals, the significant fluorescent decreases are still sufficient to demonstrate an effective switching function. By introducing more recognition regions, the switching function can be implemented by adding two inputs at the same time. This study represents the combination of the structural and functional aspects of thiolated DNA, which can serve as both a specific switching controller and a binding bridge. The strategy reported in this work can also be applied to construct more complicated molecular logic systems. We envision that the thiolated DNA participating logical switching beacon will offer unique advantages and capabilities. Moreover, this logical switching beacon could be integrated with other nano-devices for molecular detection and monitoring. This research was supported by the National Natural Science Foundation of China (61272161, 61127005, 61370099 and 61133010), the National Program on Key Basic Research Project (2013CB329600), and the Programme of Introducing Talents of Discipline to Universities (B13009).
Notes and references 1 (a) J. Bath, S. J. Green and A. J. Turberfield, Angew. Chem., Int. Ed., 2005, 44, 4358; (b) P. W. K. Rothemund, Nature, 2006, 440, 297; (c) H. Yan, S. H. Park, G. Finkelstein, J. H. Reif and T. H. LaBean, Science, 2003, 301, 1882; (d) J. Zheng, J. J. Birktoft, Y. Chen, T. Wang, R. Sha, P. E. Constantinou, S. L. Ginell, C. D. Mao and N. C. Seeman, Nature, 2009, 461, 74; (e) D. Han, S. Pal, J. Nangreave, Z. Deng, Y. Liu and H. Yan, Science, 2011, 332, 342; ( f ) U. Feldkamp and C. M. Niemeyer, Angew. Chem., Int. Ed., 2006, 45, 1856. 2 (a) J. Bath and A. J. Turberfield, Nat. Nanotechnol., 2007, 2, 275; (b) Y. Ke, S. Lindsay, Y. Chang, Y. Liu and H. Yan, Science, 2008, 319, 180; (c) E. Katz and I. Willner, Angew. Chem., Int. Ed., 2004, 43, 6042; (d) R. P. Goodman, I. A. T. Schaap, C. F. Tardin, C. M. Erben, R. M. Berry, C. F. Schmidt and A. J. Turberfield, Science, 2005, 310, 1661. 3 (a) L. M. Adleman, Science, 1994, 66, 1021; (b) L. L. Qian and E. Winfree, Science, 2011, 332, 1196; (c) D. L. Ma, H. Z. He, D. S. H. Chan and C. H. Leung, Chem. Sci., 2013, 4, 3366; (d) Q. H. Liu, L. M. Wang, A. G. Frutos, A. E. Condon, R. M. Corn and L. M. Smith, Nature, 2000, 403, 175; (e) Biomolecular Information Processing-From Logic Systems to Smart Sensors and Actuators, ed. E. Katz, Wiley-VCH, 1st edn, 2012. 4 (a) P. C. Das and A. Puri, Phys. Rev. B, 2002, 65, 155416; (b) E. Dulkeith, A. C. Morteani, T. Niedereichholz, T. A. Klar, J. Feldmann, S. A. Levi, F. van ¨ller and D. I. Gittins, Phys. Rev. Lett., 2002, Veggel, D. N. Reinhoudt, M. Mo 89, 203002; (c) C. Fan, S. Wang, J. W. Hong, G. C. Bazan, K. W. Plaxco and A. J. Heeger, Proc. Natl. Acad. Sci. U. S. A., 2003, 100, 6297; (d) C. S. Yun, A. Javier, T. Jennings, M. Fisher, S. Hira, S. Peterson, B. Hopkins, N. O. Reich and G. F. Strouse, J. Am. Chem. Soc., 2005, 127, 3115. 5 (a) S. Song, Z. Liang, J. Zhang, L. Wang, G. Li and C. Fan, Angew. Chem., Int. Ed., 2009, 48, 8670; (b) D. J. Maxwell, J. R. Taylor and S. Nie, J. Am. Chem. Soc., 2002, 124, 9606; (c) J. Zhang, L. Wang, H. Zhang, F. Boey, S. Song and C. Fan, Small, 2009, 6, 201. 6 (a) M. M. Mathew, N. Dmytro, C. Marine, V. L. Daniel and G. Oleg, Nat. Mater., 2009, 8, 388; (b) F. A. Aldaye and H. F. Sleiman, Angew. Chem., Int. Ed., 2006, 118, 2262; (c) C. A. Mirkin, R. L. Letsinger, R. C. Mucic and J. J. Storhoff, Nature, 1996, 382, 607; (d) J. Sharma, R. Chhabra, A. Cheng, J. Brownell, Y. Liu and H. Yan, Science, 2009, 323, 112; (e) J. Sharma, R. Chhabra, Y. Liu, Y. Ke and H. Yan, Angew. Chem., Int. Ed., 2006, 45, 730; ( f ) N. Dmytro, M. M. Mathew, V. L. Daniel and G. Oleg, Nature, 2008, 451, 549; ( g) S. Y. Park, A. K. R. Lytton-Jean, B. Lee, S. Weigand, G. C. Schatz and C. A. Mirkin, Nature, 2008, 451, 553. 7 A. E. Prigodich, O.-S. Lee, W. L. Daniel, D. S. Seferos, G. C. Schatz and C. A. Mirkin, J. Am. Chem. Soc., 2010, 132, 10638. 8 (a) Y. C. Cao, R. Jin and C. A. Mirkin, Science, 2002, 297, 1536; (b) N. L. Rosi and C. A. Mirkin, Chem. Rev., 2005, 105, 1547; (c) H. Li and L. J. Rothberg, J. Am. Chem. Soc., 2004, 126, 10958; (d) J. Zhang, S. Song, L. Wang, D. Pan and C. Fan, Nat. Protocols, 2007, 2, 2888; (e) J. Zhang, L. Wang, D. Pan, S. Song, F. Y. Boyer, H. Zhang and C. Fan, Small, 2008, 4, 1196; ( f ) J. Liu and Y. Lu, Angew. Chem., Int. Ed., 2005, 118, 96. This journal is
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Cite this: Chem. Commun., 2013, 49, 11308 Received 4th September 2013, Accepted 9th October 2013
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A molecular logical switching beacon controlled by thiolated DNA signals† Cheng Zhang,*a Liuqing Wu,a Jing Yang,b Shi Liub and Jin Xu*a
DOI: 10.1039/c3cc46743k www.rsc.org/chemcomm
A logical switching MB is established, with an ‘‘ON/OFF’’ switching function. In this study, thiolated DNA can participate as a switching controller to regulate the fluorescent increments of other DNA input signals. Assisted by gold nanoparticles and DNA branch migration, one and two-switch systems have been achieved.
As a nanoscale building material, DNA attracts a lot of attention in various applications including assembly materials,1 molecular machines2 and nano-computing.3 Particularly, DNA computing is an emerging research focusing on implementing both logic control and molecular detection. Lots of applications require various advanced methods to monitor or detect molecular outputs by DNA computing systems. One important class of logic computing systems is composed of gold nanoparticles (AuNPs) functionalized with DNA, taking advantage of the abilities of fluorescence quenching and aggregation.4 In fact, AuNP-based molecular beacons (MBs) possess several advantages, such as strong quenching effects, multi-functional methods and sensitive sequence specificity.5 Consequently, different approaches to construct AuNP-based MB are investigated, in which a method of attaching thiolated DNA onto the gold surface is widely used.6 For example, a multicolor nanoscale MB was constructed by Fan and co-workers, using three kinds of thiolated single strand DNA (ssDNA).5a In 2010, Mirkin’s group reported a MB to increase the rate of target hybridization based on an attachment of thiolated ssDNA.7 In previous studies about MBs, the thiol-group modified DNA is used only as a building material to bind DNA onto gold nanoparticles. In addition, triggering input signals for MBs have been limited to nucleic acids, proteins and a few small molecules.8 In order to make the thiol-group as a recognizable signal, it is necessary to establish a relationship between the attachment of the thiol-group and specific output signals. a
Institute of Software, School of Electronics Engineering and Computer Science, Peking University, Key laboratory of High Confidence Software Technologies, Ministry of Education, Beijing, China. E-mail: [email protected], [email protected] b School of Control and Computer Engineering, North China Electric Power University, Beijing, China. Fax: +86-10-61771050; Tel: +86-10-61771050 † Electronic supplementary information (ESI) available: Materials, methods, and experimental details. See DOI: 10.1039/c3cc46743k
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Fig. 1 Principles of switching MB a-B and a-Bs in (a) and (b). A logic diagram is illustrated in (c). Switching results are shown by comparing the values of Q1 and Q2 in (d).
Therefore, it is interesting to explore the possibility of using the thiol-groups as triggers to achieve molecular switching functions. In this study, we prepared a logical switching MB triggered by thiolated DNA inputs. In the MB system, corresponding fluorescent increments can be influenced by an ‘‘ON/OFF’’ switching function, where DNA hairpins B and Bs play the role of the respective triggering signals (Fig. 1). In the ‘‘ON’’ state, treated with thiol-free hairpin B, all DNA inputs can specifically cause an increase of fluorescent signals, without any hindrance. While, in the ‘‘OFF’’ state, treated with thiolated hairpin Bs, the triggering fluorescent increments can be greatly inhibited. In this system, thiolated DNA can participate as a switching controller to regulate the fluorescent increments of DNA inputs. In the first switching MB, the a mode, triggers hairpin B or Bs can hybridize with the thiolated strand A, and the DNA assembly structures can attach onto the surface of AuNPs. Then, assembly structures on MB a-B (‘‘ON’’ state) keep a ‘‘standing’’ state (Fig. 1a), while those on MB a-Bs (‘‘OFF’’ state) slightly change their gestures into a ‘‘lying’’ state (Fig. 1b). It should be noted that hairpin Bs attaches onto AuNPs not only by hybridizing with DNA strand A, but also by a connection of a thiol-group. The hybridization brings the fluorophore (labelled on DNA C-F) into the proximity of the AuNP surface, leading to a significant quenching effect. This journal is
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For the ‘‘ON’’ state, two DNA input signals D and C-z were added into a solution of MB a-B. In the presence of signal D, the assembly structure of hairpin B and C-F can be potentially displaced from AuNPs, leading to an increase of fluorescent signals. In addition, adding input signal C-z can totally displace fluorescent DNA C-F for a longer complementary hybridizing region, thereby also causing an increment of fluorescent signals. In comparison to the ‘‘ON’’ state, for the ‘‘OFF’’ state, adding input signal C-z also leads to an increase in fluorescence. When it comes to addition of signal D, although the displacement happened with a separation between hairpin Bs and strand A, the fluorophore was still in close proximity to AuNPs, because of another connection of the thiol-group from hairpin Bs. Thus, in the case of the ‘‘OFF’’ state, a significant inhibiting effect on the increase in fluorescence should be produced when signal D is added. In other words, in the logic MB a, a switching function for signal D can be achieved by adding thiol-free DNA (‘‘ON’’) or thiolated DNA (‘‘OFF’’). (NOTE: 1) The fluorescence increments were normalized as I = DF/F0, where F0 is the fluorescent intensity in the initial state without adding input strands; DF is the increment of the fluorescence, and average values are obtained by repeating the experiments three times (see ESI†). (2) Taking Imax as a reference, obtained by adding input C-z for a direct and complete displacement, the switching inhibiting effect can be judged by comparing the relative value Q = Iinput/Imax, where Iinput was obtained by adding input D or G. As demonstrated in Fig. 1a, in the ‘‘ON’’ state, in the presence of signals D and C-z, almost the same fluorescence enhancements were observed as I1 = 97.6% and I2 = 95%, respectively (Q1 = 1.02 : 1). However, in the ‘‘OFF’’ state, the fluorescent increment from signal D (I3 = 84.3%) was much less than that of the complete displacement from C-z (I4 = 163.5%) (Q2 = 0.52 : 1). Compared with values of Q1, a great decrease of Q2 may suggest an existence of inhibiting effects in the ‘‘OFF’’ state. To further test whether the switching system can be scaled up to a two-switch system, the second switching MB, the b mode was employed, where an additional displacing site, capable of recognizing the input strand G, was introduced (Fig. 2). In the second switching MB b mode, the DNA assembly structure consists of three strands, named AD, AC and hairpin B/Bs. After hybridizing with fluorescent
DNA C-F, the MB can be triggered by three input signals: D, G and C-z. For the ‘‘ON’’ state (with hairpin B), in the presence of either input signal D or G, the fluorescent DNA complex will dissociate from the AuNPs, thus causing increments of fluorescent signals. On the other hand, treating the MB system with hairpin Bs yields the ‘‘OFF’’ state, where a connection between a thiol-group (hairpin Bs) and an AuNP can be generated. In this case, no matter whether input D or G is added, the fluorescent DNA complex will still attach onto the surface of the AuNPs. Therefore, a significant inhibiting effect on fluorescent enhancement should be produced. In Fig. 2, it can be observed that in the ‘‘ON’’ state, addition of three inputs D, G and C-z has approximately increasing efficiencies of 58.8%, 53% and 55.9%, respectively (Q3 = 1.05 : 0.95 : 1). Almost no difference could be observed among addition of D, G and C-z. While, in the ‘‘OFF’’ state, the fluorescent increments upon adding inputs D and G are 39.2% and 39.2%, respectively, which are much lower than 104.1% upon adding input C-z (Q4 = 0.38 : 0.38 : 1). Compared with Q3 and Q4, the results suggest that a significant inhibiting effect can be induced by the participation of the thiolated DNA signals. It should be noted that, despite having the inhibiting effect in the ‘‘OFF’’ state, the fluorescent increments still existed when adding input strands, suggesting that not all connections were generated between thiolated hairpin Bs and AuNPs. To explore more details of the inhibiting effects induced by thiol-group attachment, it is important to consider efficiencies of such attachment, influenced by structural changes. Fig. 3 shows a schematic diagram of two different ways of attachment with a ‘‘blunt’’ end and a ‘‘protruding’’ end. Here, two kinds of hairpin DNA Bs and B1s are utilized to form two systems: MB b-Bs and MB b-B1s, respectively. In the case of the ‘‘protruding’’ end, the thiolgroup is connected with a flexible ssDNA arm, which is a favorable structure for the attachment. While in the case of the ‘‘blunt’’ end, although the thiol-group is exposed to interact with the surface of the AuNPs, the spatial hindrance of the blunt end of the ssDNA may still impede the attachment of the thiol-group. Using the flexible ‘‘protruding’’ arm, in the ‘‘OFF’’ state, adding inputs D, G and C-z leads to the corresponding fluorescent increments of 57.7%, 56.1% and 60.03% (Q5 = 0.96 : 0.93 : 1). In the ‘‘ON’’
Fig. 2 Principles of switching MB b-B and b-Bs in (a) and (b). A logic diagram is illustrated in (c). Switching results are shown by comparing the values of Q3 and Q4 in (d).
Fig. 3 A schematic diagram of two different ways of attachment with the ‘‘blunt’’ end and the ‘‘protruding’’ end in (a) and (b). Switching results are shown by comparing the values of Q5 and Q6 in (c).
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Fig. 4 Comparisons of the MB b structures of mono-functionalized conjugates (a). Electrophoretic results of 5 nm AuNPs are displayed in 3% agarose gel. (b) MB a-B was purified, and then displaced by adding trigger strands: D (Lane 3) and G (Lane 4). (c) An assembly of a discrete AuNP cluster (5 + 15 nm nanoparticles), and its TEM images.
state of the ‘‘protruding’’ arm, the experimental data were 20.37%, 20.23% and 203.13% (Q6 = 0.1 : 0.1 : 1). Comparing Q4 = 0.38 : 0.38 : 1 and Q6 = 0.1 : 0.1 : 1, it is easy to see that using the ‘‘protruding’’ end has significantly greater inhibiting effects than using the ‘‘blunt’’ end. This phenomenon may be due to that, although the ‘‘blunt’’ end thiol-groups were in the proximity of AuNPs, it is rather difficult for all of them to attach onto the surface of AuNPs because of the strong spatial hindrance. Therefore, the displacements induced by adding inputs D and G will lead to some separation between the fluorophore and AuNPs, and some fluorescent increments will be produced. However, for the ‘‘protruding’’ end, almost all of the thiol-groups were favored to attach onto AuNPs because of their flexible arms, leading to a significant inhibiting effect. Thus, adding inputs D and G will cause a much less fluorescent increment, when compared with using the ‘‘blunt’’ end. In this work, the triggering process of adding input DNA can be confirmed by comparing the MB b structures of mono-functionalized conjugates. By adding different DNA assembly structures: AD, AD + AC, AD + AC + B1 and AD + AC + B1s, the corresponding conjugates with various gel running speeds were generated as shown in Fig. 4a. As expected, the bands of conjugating products shifted slowly as the complexities of DNA assembly structures were increased. Interestingly, we investigated mono-functionalized conjugate MB b-B1 using DNA branch migration, and results of the displacing products in the gel are illustrated (Fig. 4b). As a comparison, MB a-B was purified and displaced by adding strands: D (Lane 3) and G (Lane 4). The increasing gel running speeds of the displacing products suggested that the DNA structures left the AuNPs. In addition, to test whether the bulged DNA loop of hairpin B1 can provide a binding site for further detection, a method of assembly of discrete AuNP clusters was employed. The addition of 5 nm multivalent conjugate B1NP led to a cooperative hybridization with hairpin B1, resulting in the formation of a dimer of gold nanoparticles with asymmetric diameters, 5 and 15 nm (Fig. 4c). In these experiments, we demonstrated the mono-functionalized MB b to be a well-formed conjugate, containing a unique hybridizing site and two triggering toehold regions for strand displacement. In this communication, we present a logical MB system controlled by thiolated DNA input signals, which allows achieving molecular switching functions. Although the ‘‘OFF’’ state was not 11310
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Communication able to completely inhibit enhancement of fluorescent signals, the significant fluorescent decreases are still sufficient to demonstrate an effective switching function. By introducing more recognition regions, the switching function can be implemented by adding two inputs at the same time. This study represents the combination of the structural and functional aspects of thiolated DNA, which can serve as both a specific switching controller and a binding bridge. The strategy reported in this work can also be applied to construct more complicated molecular logic systems. We envision that the thiolated DNA participating logical switching beacon will offer unique advantages and capabilities. Moreover, this logical switching beacon could be integrated with other nano-devices for molecular detection and monitoring. This research was supported by the National Natural Science Foundation of China (61272161, 61127005, 61370099 and 61133010), the National Program on Key Basic Research Project (2013CB329600), and the Programme of Introducing Talents of Discipline to Universities (B13009).
Notes and references 1 (a) J. Bath, S. J. Green and A. J. Turberfield, Angew. Chem., Int. Ed., 2005, 44, 4358; (b) P. W. K. Rothemund, Nature, 2006, 440, 297; (c) H. Yan, S. H. Park, G. Finkelstein, J. H. Reif and T. H. LaBean, Science, 2003, 301, 1882; (d) J. Zheng, J. J. Birktoft, Y. Chen, T. Wang, R. Sha, P. E. Constantinou, S. L. Ginell, C. D. Mao and N. C. Seeman, Nature, 2009, 461, 74; (e) D. Han, S. Pal, J. Nangreave, Z. Deng, Y. Liu and H. Yan, Science, 2011, 332, 342; ( f ) U. Feldkamp and C. M. Niemeyer, Angew. Chem., Int. Ed., 2006, 45, 1856. 2 (a) J. Bath and A. J. Turberfield, Nat. Nanotechnol., 2007, 2, 275; (b) Y. Ke, S. Lindsay, Y. Chang, Y. Liu and H. Yan, Science, 2008, 319, 180; (c) E. Katz and I. Willner, Angew. Chem., Int. Ed., 2004, 43, 6042; (d) R. P. Goodman, I. A. T. Schaap, C. F. Tardin, C. M. Erben, R. M. Berry, C. F. Schmidt and A. J. Turberfield, Science, 2005, 310, 1661. 3 (a) L. M. Adleman, Science, 1994, 66, 1021; (b) L. L. Qian and E. Winfree, Science, 2011, 332, 1196; (c) D. L. Ma, H. Z. He, D. S. H. Chan and C. H. Leung, Chem. Sci., 2013, 4, 3366; (d) Q. H. Liu, L. M. Wang, A. G. Frutos, A. E. Condon, R. M. Corn and L. M. Smith, Nature, 2000, 403, 175; (e) Biomolecular Information Processing-From Logic Systems to Smart Sensors and Actuators, ed. E. Katz, Wiley-VCH, 1st edn, 2012. 4 (a) P. C. Das and A. Puri, Phys. Rev. B, 2002, 65, 155416; (b) E. Dulkeith, A. C. Morteani, T. Niedereichholz, T. A. Klar, J. Feldmann, S. A. Levi, F. van ¨ller and D. I. Gittins, Phys. Rev. Lett., 2002, Veggel, D. N. Reinhoudt, M. Mo 89, 203002; (c) C. Fan, S. Wang, J. W. Hong, G. C. Bazan, K. W. Plaxco and A. J. Heeger, Proc. Natl. Acad. Sci. U. S. A., 2003, 100, 6297; (d) C. S. Yun, A. Javier, T. Jennings, M. Fisher, S. Hira, S. Peterson, B. Hopkins, N. O. Reich and G. F. Strouse, J. Am. Chem. Soc., 2005, 127, 3115. 5 (a) S. Song, Z. Liang, J. Zhang, L. Wang, G. Li and C. Fan, Angew. Chem., Int. Ed., 2009, 48, 8670; (b) D. J. Maxwell, J. R. Taylor and S. Nie, J. Am. Chem. Soc., 2002, 124, 9606; (c) J. Zhang, L. Wang, H. Zhang, F. Boey, S. Song and C. Fan, Small, 2009, 6, 201. 6 (a) M. M. Mathew, N. Dmytro, C. Marine, V. L. Daniel and G. Oleg, Nat. Mater., 2009, 8, 388; (b) F. A. Aldaye and H. F. Sleiman, Angew. Chem., Int. Ed., 2006, 118, 2262; (c) C. A. Mirkin, R. L. Letsinger, R. C. Mucic and J. J. Storhoff, Nature, 1996, 382, 607; (d) J. Sharma, R. Chhabra, A. Cheng, J. Brownell, Y. Liu and H. Yan, Science, 2009, 323, 112; (e) J. Sharma, R. Chhabra, Y. Liu, Y. Ke and H. Yan, Angew. Chem., Int. Ed., 2006, 45, 730; ( f ) N. Dmytro, M. M. Mathew, V. L. Daniel and G. Oleg, Nature, 2008, 451, 549; ( g) S. Y. Park, A. K. R. Lytton-Jean, B. Lee, S. Weigand, G. C. Schatz and C. A. Mirkin, Nature, 2008, 451, 553. 7 A. E. Prigodich, O.-S. Lee, W. L. Daniel, D. S. Seferos, G. C. Schatz and C. A. Mirkin, J. Am. Chem. Soc., 2010, 132, 10638. 8 (a) Y. C. Cao, R. Jin and C. A. Mirkin, Science, 2002, 297, 1536; (b) N. L. Rosi and C. A. Mirkin, Chem. Rev., 2005, 105, 1547; (c) H. Li and L. J. Rothberg, J. Am. Chem. Soc., 2004, 126, 10958; (d) J. Zhang, S. Song, L. Wang, D. Pan and C. Fan, Nat. Protocols, 2007, 2, 2888; (e) J. Zhang, L. Wang, D. Pan, S. Song, F. Y. Boyer, H. Zhang and C. Fan, Small, 2008, 4, 1196; ( f ) J. Liu and Y. Lu, Angew. Chem., Int. Ed., 2005, 118, 96. This journal is
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