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Molecular aptamer beacon tuned DNA strand displacement to transform small molecules into DNA logic outputs†
Received 30th December 2013, Accepted 4th February 2014
Jinbo Zhu,ab Libing Zhang,ab Zhixue Zhou,ab Shaojun Dongab and Erkang Wang*ab
DOI: 10.1039/c3cc49833f www.rsc.org/chemcomm
A molecular aptamer beacon tuned DNA strand displacement reaction was introduced in this work. This strand displacement mode can be used to transform the adenosine triphosphate (ATP) input into a DNA strand output signal for the downstream gates to process. A simple logic circuit was built on the basis of this mechanism.
Molecular computing is an emerging interdisciplinary area. Research studies on its theory, operation and applications are frequently reported, especially on DNA computing systems.1 The theoretical possibility of a DNA computer in intelligent diagnosis and therapy of cancer has drawn widespread attention.2 The information is usually carried and transferred by DNA strands among the logic gates in most DNA computing systems. It is easy to receive the input signal in the form of a DNA strand for the logic devices made from DNA materials, but it is still a challenge in other molecular forms. Therefore, for applying a DNA computer in a complex biosystem, it is necessary to design a versatile sensing platform that can transform various input molecular signals into DNA strands to be processed by the DNA computer. DNA aptamers are able to bind to various molecular targets such as proteins, small molecules, metal ions and even cells.3 Using aptamers as receptors to translate multiform input signals into DNA strands as outputs will broaden the range of sensible input types of the DNA computer.4 A molecular beacon is a hairpin shaped molecule with an internally quenched fluorophore, whose fluorescence is restored when it binds to a target nucleic acid strand.5 By combining a molecular beacon with the aptamer technology, a molecular aptamer beacon (MAB) has been introduced and used to detect a variety of biomolecules.6 Since an MAB can interact with target molecules
a
State Key Laboratory of Electroanalytical Chemistry, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun, Jilin 130022, China. E-mail: [email protected]; Fax: +86-431-85689711; Tel: +86-431-85262101 b University of Chinese Academy of Sciences, Beijing, 100049, China † Electronic supplementary information (ESI) available: Experimental details and supplementary figures. See DOI: 10.1039/c3cc49833f
This journal is © The Royal Society of Chemistry 2014
and result in the change in DNA conformation, it would be an ideal candidate for the receptor of a DNA computer to sense various inputs. A toehold-mediated strand displacement reaction (SDR) is an important tool in DNA computing to receive and process the input strand signals. It has been widely used in constructing logic gates, circuits and even neural networks.7 Herein, we utilized an MAB to tune the toehold-mediated DNA SDR, and then built a new sensing platform based on it, which can transform small molecules into DNA logic outputs. ATP and its aptamer were taken as a model in this work.8 The ATP binding aptamer (ABA) sequence was assembled into a hairpin structure. The toehold domain was located in the stem of the hairpin and hybridized with its complementary sequence. After binding to ATP, the conformation of the hairpin changed and the toehold domain was released from the double strand. The mechanism of the MAB tuned SDR has been successfully verified by native polyacrylamide gel electrophoresis (PAGE). To facilitate the applications of this system, a label-free signal readout strategy based on split G-quadruplex enhanced protoporphyrin IX (PPIX) fluorescence was used to give off the final readable fluorescence signal.9 This sensing platform could work as a biosensor for label-free detection of ATP and DNA. Furthermore, a simple logic circuit was built based on this molecular system with ATP and DNA as inputs. By altering the aptamer sequence it will be able to receive more stimuli. There is a good prospect of this molecule platform in biosensors and molecular computing. The schematic diagram is shown in Fig. 1a. Strand M is the main component of this device. The pink loop part is made up of the ATP aptamer sequence, which takes the responsibility of receiving the input ATP signal. The green part is the toehold domain, which is blocked by its complementary sequence in the hairpin structure. The red part of M is used to bind the output strand S to form the S–M complex. If the toehold domain is exposed in solution, strand R will displace the S strand in the S–M complex to form the R–M complex through the toehold. After ATP is added into this system, this small
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Fig. 1 (a) ATP and the cABA interact with the MAB to tune the strand displacement reaction. Different functional domains are plotted in different colors. (b) Fluorescence emission spectra of the complexes of PPIX and different DNAs: (a) G1, G2, S; (b) G1, G2, S, M; (c) G1, G2, S, M, ATP; (d) G1, G2, S, M, cABA; (e) G1, G2, S, M, R; (f) G1, G2, S, M, R, cABA; (g) G1, G2, S, M, R, ATP. The fluorescent analysis was performed in the TEK buffer with a final concentration of 1.2 mM for PPIX, 0.16 mM for G1 and G2, 192 mM for ATP and 0.1 mM for the other strands. (c) Dependence of the normalized FI at 630 nm of the sensing system (PPIX, G1, G2, S, M, R) on the ATP concentration from 32 to 192 mM. The data were collected from three independent experiments. Fluorescence emission spectra are shown in Fig. S1 (ESI†).
molecule will bind to its aptamer in the loop. The binding between ATP and the ABA disbands the hairpin structure. The double stranded part of this stem-loop structure is opened and the toehold domain is exposed to R in solution. With the assistance of the toehold, R hybridizes with M and displaces S. Two G-rich segments, G1 and G2, are designed to capture the output strand from the upstream gate. When free S is present, G1 and G2 will be drawn together to form a split G-quadruplex structure with S through hybridization. The split G-quadruplex can specifically bind PPIX and dramatically enhance its fluorescence. The fluorescence results are shown in Fig. 1b. When S was blocked by M, the fluorescence intensity (FI) was very low and was little affected by the addition of ATP (curves b and c). Although R was added, the FI was still kept at a low level (curve e), which demonstrated that R alone was insufficient to displace S without the help of the free toehold. Only when both R and ATP were present, was S given off and an increase of fluorescence was induced (curve g). The more ATP was added, the more strand S was given off. The amount of the split G-quadruplex also increased with the addition of S, hence the excess PPIX in solution could bind to the newly formed split G-quadruplexes, and higher fluorescence was given. As shown in Fig. 1c and Fig. S1 (ESI†), the FI increased with the addition of ATP, and a linear relationship between FI and the ATP concentration was identified in the range from 32 to 192 mM with a detection limit of 25 mM (three times the standard deviation of the blank solution). To test the selectivity of this ATP aptasensor, the other nucleosides (CTP, GTP and UTP) were adopted in place of ATP. The results in Fig. S2 (ESI†) proved the outstanding selectivity of this sensor for ATP detection. Since molecular beacons were originally used to detect DNA, similarly, this MAB based sensing platform can also effortlessly receive the DNA input signal. The schematic diagram is also
3322 | Chem. Commun., 2014, 50, 3321--3323
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illustrated in Fig. 1a. The complementary strand of ABA, cABA, can hybridize with the loop of M to disband the hairpin structure and provide the free toehold for R. The subsequent reactions are the same as the ones initiated by ATP. The fluorescence results are shown in Fig. 1b. The fluorescence was restored to a high level in the presence of both the cABA and R (Fig. 1b, curve f). Therefore, this molecular platform can also be regarded as a special label-free molecular beacon to detect the DNA strand cABA. Results in Fig. S3 (ESI†) show that the fluorescence intensity increased with the addition of the cABA and a linear relationship between the FI at 630 nm and the cABA concentration was identified from 20 to 120 nM with a detection limit of 11 nM (three times the standard deviation of the blank solution). Importantly, it can be easily changed according to the target strand by altering the sequence of the loop domain. The mechanism of this MAB tuned DNA SDR was verified by native PAGE (Fig. 2). The added DNA strands in different lanes are indicated in the table. When compared with the bands in lanes 1 and 3, the single band in lane 5 proved the hybridization between S and M. Addition of R in lane 6 had little effect on the stability of the S–M complex, which proved that the toehold domain in the stem part is well protected by the hairpin structure. However, it is worth noting that a new weak band emerged in lane 6, which can be ascribed to the formation of the R–M complex. The electrophoretic mobility of the bands got higher with the decrease of length and weight of DNA strands. Since R is shorter than S, the band of the R–M complex had a higher mobility than that of the S–M complex. Formation of the weak band of the R–M complex indicated that a small part of strand S on M was replaced by R (since the amount of released free S was very small, we could not observe a clear band of S in lane 6), but most of S still hybridized with M and the corresponding FI was indeed still kept at a low level (Fig. 1b, curve e). Effects of input ATP and cABA for this system were investigated and results are shown in lanes 7–10. Due to the small weight of ATP, the band of the S–M–ATP complex formed in lane 10 was
Fig. 2 Native 15% polyacrylamide gel analysis of the MAB tuned DNA SDR. DNA strands added in every lane are indicated in the table. The concentration of each DNA in PAGE is 0.2 mM. ATP is added at a concentration of 3.2 mM.
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at the same position as that of the S–M complex. In lane 9, when both ATP and R were added, the band of S could be observed, the band of the R–M–ATP complex appeared at the same position as that of the R–M complex, and the band of the S–M–ATP complex became very weak. This phenomenon indicated that most of S had been displaced by R in the DNA complex. However, without the mediation of ATP, the SDR could not proceed smoothly in lane 6, which confirmed the vital position of ATP in this reaction. Similar results were observed in lanes 7 and 8. The band with the smallest shift in lane 8 can be ascribed to the complex of S, M and cABA. After the addition of R, the band of S appeared and a band with a higher mobility compared with the band of S–M–cABA formed in lane 7, which can be attributed to the formation of the R–M–cABA complex. Fading of the band of the S–M–cABA complex and appearance of the bands of the S and R–M–cABA complex in lane 7 demonstrated that the strand displacement reaction was carried out effectively with the help of the cABA. Based on the properties of this MAB tuned SDR, it can be applied to build a simple logic circuit to receive the ATP and DNA inputs. The circuit and its truth table are shown in Fig. 3a and Fig. S4 (ESI†), respectively. The MAB based sensing platform is the center of this circuit in which it works as an AND gate. Since ATP and cABA can both open the loop of the S–M complex to expose the toehold domain for R, they can be seen as two inputs of an OR gate in this circuit. Either one or both of them can cooperate with R to release strand S in the S–M complex as an output of the AND gate. The output free strand S will draw the two G-rich segments to form the split G-quadruplex, which can dramatically enhance the fluorescence of PPIX. This is a simple YES gate that translates the DNA strand output
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signal into the fluorescence signal for readout. The normalized fluorescence results for different inputs are shown in Fig. 3b. As expected the results are in good agreement with our design. The primary value of this circuit is that it can transform the ATP input into a DNA strand output signal; thus it can be accepted and processed smoothly by the downstream circuits. Fabrication of this ATP tuned aptamer-based DNA logic circuit demonstrates the application prospect of the DNA computer in biosystems. In summary, an MAB tuned DNA SDR has been introduced in this work. Based on this mechanism, a new sensing platform was constructed, which can receive the small molecule input and transform it into a DNA strand output signal for downstream gates to process. Because of the nature of the molecular beacon, the DNA strand input can also be received by the sensing platform to tune the logic circuit. The mechanism of the MAB tuned SDR was identified through PAGE. Split G-quadruplex enhanced fluorescence of PPIX was used to report the final output signal. This molecular system can also work as a label-free biosensor for ATP and DNA detection. Furthermore, we built a simple logic circuit based on this SDR mode with ATP and DNA strands as input. Incorporating an aptamer sequence into the DNA logic system and applying small molecules as input to tune the output signal of the logic circuit are very significant for the application of the DNA computing system in a wide range of fields. Realizing a disease marker sensitive smart drug delivery system will be quite promising if some relationship between the final output signal and drug release is established. This work was supported by the National Natural Science Foundation of China (Grants 21075116 and 21190040) and 973 projects (2010CB933600 and 2011CB911000).
Notes and references
Fig. 3 (a) Schematic diagram of the logic circuit with ATP and DNA strands as inputs. (b) Normalized FI at 630 nm of the different input modes of the circuit. FI at 630 nm of curve a in Fig. 1b is set as 1. Threshold value is set at 0.4 to judge the positive and negative output signals. The three binary numbers represent the input status of ATP, cABA and R, respectively. The data were collected from three independent experiments.
This journal is © The Royal Society of Chemistry 2014
1 (a) A. J. Ruben and L. F. Landweber, Nat. Rev. Mol. Cell Biol., 2000, 1, 69; (b) E. Katz and V. Privman, Chem. Soc. Rev., 2010, 39, 1835; (c) L. Qian and E. Winfree, Science, 2011, 332, 1196; (d) J. Zhu, T. Li, L. Zhang, S. Dong and E. Wang, Biomaterials, 2011, 32, 7318. 2 (a) Y. Benenson, Nat. Rev. Genet., 2012, 13, 455; (b) E. Shapiro and Y. Benenson, Sci. Am., 2006, 294, 44; (c) D. Han, G. Zhu, C. Wu, Z. Zhu, T. Chen, X. Zhang and W. Tan, ACS Nano, 2013, 2312. 3 (a) W. Tan, M. J. Donovan and J. Jiang, Chem. Rev., 2013, 2842; (b) J. Liu and Y. Lu, Angew. Chem., Int. Ed., 2007, 46, 7587. 4 (a) D. Han, Z. Zhu, C. Wu, L. Peng, L. Zhou, B. Gulbakan, G. Zhu, K. R. Williams and W. Tan, J. Am. Chem. Soc., 2012, 134, 20797; (b) Y. Xing, Z. Yang and D. Liu, Angew. Chem., Int. Ed., 2011, 50, 11934; (c) J. Zhu, L. Zhang, Z. Zhou, S. Dong and E. Wang, Anal. Chem., 2014, 86, 312. 5 W. H. Tan, K. M. Wang and T. J. Drake, Curr. Opin. Chem. Biol., 2004, 8, 547. 6 (a) Z. Cai, Y. Song, Y. Wu, Z. Zhu, C. James Yang and X. Chen, Biosens. Bioelectron., 2013, 41, 783; (b) X. Tan, W. Chen, S. Lu, Z. Zhu, T. Chen, G. Zhu, M. You and W. Tan, Anal. Chem., 2012, 84, 8272. 7 (a) G. Seelig, D. Soloveichik, D. Y. Zhang and E. Winfree, Science, 2006, 314, 1585; (b) L. Qian, E. Winfree and J. Bruck, Nature, 2011, 475, 368; (c) J. Zhu, L. Zhang, T. Li, S. Dong and E. Wang, Adv. Mater., 2013, 25, 2440; (d) J. Zhu, L. Zhang, S. Dong and E. Wang, ACS Nano, 2013, 7, 10211. 8 (a) S. Guo, Y. Du, X. Yang, S. Dong and E. Wang, Anal. Chem., 2011, 83, 8035; (b) Y. Xiang, X. Q. Qian, Y. Y. Zhang, Y. Chen, Y. Q. Chai and R. Yuan, Biosens. Bioelectron., 2011, 26, 3077. 9 (a) J. Zhu, L. Zhang and E. Wang, Chem. Commun., 2012, 48, 11990; (b) T. Li, E. Wang and S. Dong, Anal. Chem., 2010, 82, 7576.
Chem. Commun., 2014, 50, 3321--3323 | 3323
COMMUNICATION
View Article Online View Journal | View Issue
Cite this: Chem. Commun., 2014, 50, 3321
Molecular aptamer beacon tuned DNA strand displacement to transform small molecules into DNA logic outputs†
Received 30th December 2013, Accepted 4th February 2014
Jinbo Zhu,ab Libing Zhang,ab Zhixue Zhou,ab Shaojun Dongab and Erkang Wang*ab
DOI: 10.1039/c3cc49833f www.rsc.org/chemcomm
A molecular aptamer beacon tuned DNA strand displacement reaction was introduced in this work. This strand displacement mode can be used to transform the adenosine triphosphate (ATP) input into a DNA strand output signal for the downstream gates to process. A simple logic circuit was built on the basis of this mechanism.
Molecular computing is an emerging interdisciplinary area. Research studies on its theory, operation and applications are frequently reported, especially on DNA computing systems.1 The theoretical possibility of a DNA computer in intelligent diagnosis and therapy of cancer has drawn widespread attention.2 The information is usually carried and transferred by DNA strands among the logic gates in most DNA computing systems. It is easy to receive the input signal in the form of a DNA strand for the logic devices made from DNA materials, but it is still a challenge in other molecular forms. Therefore, for applying a DNA computer in a complex biosystem, it is necessary to design a versatile sensing platform that can transform various input molecular signals into DNA strands to be processed by the DNA computer. DNA aptamers are able to bind to various molecular targets such as proteins, small molecules, metal ions and even cells.3 Using aptamers as receptors to translate multiform input signals into DNA strands as outputs will broaden the range of sensible input types of the DNA computer.4 A molecular beacon is a hairpin shaped molecule with an internally quenched fluorophore, whose fluorescence is restored when it binds to a target nucleic acid strand.5 By combining a molecular beacon with the aptamer technology, a molecular aptamer beacon (MAB) has been introduced and used to detect a variety of biomolecules.6 Since an MAB can interact with target molecules
a
State Key Laboratory of Electroanalytical Chemistry, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun, Jilin 130022, China. E-mail: [email protected]; Fax: +86-431-85689711; Tel: +86-431-85262101 b University of Chinese Academy of Sciences, Beijing, 100049, China † Electronic supplementary information (ESI) available: Experimental details and supplementary figures. See DOI: 10.1039/c3cc49833f
This journal is © The Royal Society of Chemistry 2014
and result in the change in DNA conformation, it would be an ideal candidate for the receptor of a DNA computer to sense various inputs. A toehold-mediated strand displacement reaction (SDR) is an important tool in DNA computing to receive and process the input strand signals. It has been widely used in constructing logic gates, circuits and even neural networks.7 Herein, we utilized an MAB to tune the toehold-mediated DNA SDR, and then built a new sensing platform based on it, which can transform small molecules into DNA logic outputs. ATP and its aptamer were taken as a model in this work.8 The ATP binding aptamer (ABA) sequence was assembled into a hairpin structure. The toehold domain was located in the stem of the hairpin and hybridized with its complementary sequence. After binding to ATP, the conformation of the hairpin changed and the toehold domain was released from the double strand. The mechanism of the MAB tuned SDR has been successfully verified by native polyacrylamide gel electrophoresis (PAGE). To facilitate the applications of this system, a label-free signal readout strategy based on split G-quadruplex enhanced protoporphyrin IX (PPIX) fluorescence was used to give off the final readable fluorescence signal.9 This sensing platform could work as a biosensor for label-free detection of ATP and DNA. Furthermore, a simple logic circuit was built based on this molecular system with ATP and DNA as inputs. By altering the aptamer sequence it will be able to receive more stimuli. There is a good prospect of this molecule platform in biosensors and molecular computing. The schematic diagram is shown in Fig. 1a. Strand M is the main component of this device. The pink loop part is made up of the ATP aptamer sequence, which takes the responsibility of receiving the input ATP signal. The green part is the toehold domain, which is blocked by its complementary sequence in the hairpin structure. The red part of M is used to bind the output strand S to form the S–M complex. If the toehold domain is exposed in solution, strand R will displace the S strand in the S–M complex to form the R–M complex through the toehold. After ATP is added into this system, this small
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Fig. 1 (a) ATP and the cABA interact with the MAB to tune the strand displacement reaction. Different functional domains are plotted in different colors. (b) Fluorescence emission spectra of the complexes of PPIX and different DNAs: (a) G1, G2, S; (b) G1, G2, S, M; (c) G1, G2, S, M, ATP; (d) G1, G2, S, M, cABA; (e) G1, G2, S, M, R; (f) G1, G2, S, M, R, cABA; (g) G1, G2, S, M, R, ATP. The fluorescent analysis was performed in the TEK buffer with a final concentration of 1.2 mM for PPIX, 0.16 mM for G1 and G2, 192 mM for ATP and 0.1 mM for the other strands. (c) Dependence of the normalized FI at 630 nm of the sensing system (PPIX, G1, G2, S, M, R) on the ATP concentration from 32 to 192 mM. The data were collected from three independent experiments. Fluorescence emission spectra are shown in Fig. S1 (ESI†).
molecule will bind to its aptamer in the loop. The binding between ATP and the ABA disbands the hairpin structure. The double stranded part of this stem-loop structure is opened and the toehold domain is exposed to R in solution. With the assistance of the toehold, R hybridizes with M and displaces S. Two G-rich segments, G1 and G2, are designed to capture the output strand from the upstream gate. When free S is present, G1 and G2 will be drawn together to form a split G-quadruplex structure with S through hybridization. The split G-quadruplex can specifically bind PPIX and dramatically enhance its fluorescence. The fluorescence results are shown in Fig. 1b. When S was blocked by M, the fluorescence intensity (FI) was very low and was little affected by the addition of ATP (curves b and c). Although R was added, the FI was still kept at a low level (curve e), which demonstrated that R alone was insufficient to displace S without the help of the free toehold. Only when both R and ATP were present, was S given off and an increase of fluorescence was induced (curve g). The more ATP was added, the more strand S was given off. The amount of the split G-quadruplex also increased with the addition of S, hence the excess PPIX in solution could bind to the newly formed split G-quadruplexes, and higher fluorescence was given. As shown in Fig. 1c and Fig. S1 (ESI†), the FI increased with the addition of ATP, and a linear relationship between FI and the ATP concentration was identified in the range from 32 to 192 mM with a detection limit of 25 mM (three times the standard deviation of the blank solution). To test the selectivity of this ATP aptasensor, the other nucleosides (CTP, GTP and UTP) were adopted in place of ATP. The results in Fig. S2 (ESI†) proved the outstanding selectivity of this sensor for ATP detection. Since molecular beacons were originally used to detect DNA, similarly, this MAB based sensing platform can also effortlessly receive the DNA input signal. The schematic diagram is also
3322 | Chem. Commun., 2014, 50, 3321--3323
ChemComm
illustrated in Fig. 1a. The complementary strand of ABA, cABA, can hybridize with the loop of M to disband the hairpin structure and provide the free toehold for R. The subsequent reactions are the same as the ones initiated by ATP. The fluorescence results are shown in Fig. 1b. The fluorescence was restored to a high level in the presence of both the cABA and R (Fig. 1b, curve f). Therefore, this molecular platform can also be regarded as a special label-free molecular beacon to detect the DNA strand cABA. Results in Fig. S3 (ESI†) show that the fluorescence intensity increased with the addition of the cABA and a linear relationship between the FI at 630 nm and the cABA concentration was identified from 20 to 120 nM with a detection limit of 11 nM (three times the standard deviation of the blank solution). Importantly, it can be easily changed according to the target strand by altering the sequence of the loop domain. The mechanism of this MAB tuned DNA SDR was verified by native PAGE (Fig. 2). The added DNA strands in different lanes are indicated in the table. When compared with the bands in lanes 1 and 3, the single band in lane 5 proved the hybridization between S and M. Addition of R in lane 6 had little effect on the stability of the S–M complex, which proved that the toehold domain in the stem part is well protected by the hairpin structure. However, it is worth noting that a new weak band emerged in lane 6, which can be ascribed to the formation of the R–M complex. The electrophoretic mobility of the bands got higher with the decrease of length and weight of DNA strands. Since R is shorter than S, the band of the R–M complex had a higher mobility than that of the S–M complex. Formation of the weak band of the R–M complex indicated that a small part of strand S on M was replaced by R (since the amount of released free S was very small, we could not observe a clear band of S in lane 6), but most of S still hybridized with M and the corresponding FI was indeed still kept at a low level (Fig. 1b, curve e). Effects of input ATP and cABA for this system were investigated and results are shown in lanes 7–10. Due to the small weight of ATP, the band of the S–M–ATP complex formed in lane 10 was
Fig. 2 Native 15% polyacrylamide gel analysis of the MAB tuned DNA SDR. DNA strands added in every lane are indicated in the table. The concentration of each DNA in PAGE is 0.2 mM. ATP is added at a concentration of 3.2 mM.
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at the same position as that of the S–M complex. In lane 9, when both ATP and R were added, the band of S could be observed, the band of the R–M–ATP complex appeared at the same position as that of the R–M complex, and the band of the S–M–ATP complex became very weak. This phenomenon indicated that most of S had been displaced by R in the DNA complex. However, without the mediation of ATP, the SDR could not proceed smoothly in lane 6, which confirmed the vital position of ATP in this reaction. Similar results were observed in lanes 7 and 8. The band with the smallest shift in lane 8 can be ascribed to the complex of S, M and cABA. After the addition of R, the band of S appeared and a band with a higher mobility compared with the band of S–M–cABA formed in lane 7, which can be attributed to the formation of the R–M–cABA complex. Fading of the band of the S–M–cABA complex and appearance of the bands of the S and R–M–cABA complex in lane 7 demonstrated that the strand displacement reaction was carried out effectively with the help of the cABA. Based on the properties of this MAB tuned SDR, it can be applied to build a simple logic circuit to receive the ATP and DNA inputs. The circuit and its truth table are shown in Fig. 3a and Fig. S4 (ESI†), respectively. The MAB based sensing platform is the center of this circuit in which it works as an AND gate. Since ATP and cABA can both open the loop of the S–M complex to expose the toehold domain for R, they can be seen as two inputs of an OR gate in this circuit. Either one or both of them can cooperate with R to release strand S in the S–M complex as an output of the AND gate. The output free strand S will draw the two G-rich segments to form the split G-quadruplex, which can dramatically enhance the fluorescence of PPIX. This is a simple YES gate that translates the DNA strand output
Communication
signal into the fluorescence signal for readout. The normalized fluorescence results for different inputs are shown in Fig. 3b. As expected the results are in good agreement with our design. The primary value of this circuit is that it can transform the ATP input into a DNA strand output signal; thus it can be accepted and processed smoothly by the downstream circuits. Fabrication of this ATP tuned aptamer-based DNA logic circuit demonstrates the application prospect of the DNA computer in biosystems. In summary, an MAB tuned DNA SDR has been introduced in this work. Based on this mechanism, a new sensing platform was constructed, which can receive the small molecule input and transform it into a DNA strand output signal for downstream gates to process. Because of the nature of the molecular beacon, the DNA strand input can also be received by the sensing platform to tune the logic circuit. The mechanism of the MAB tuned SDR was identified through PAGE. Split G-quadruplex enhanced fluorescence of PPIX was used to report the final output signal. This molecular system can also work as a label-free biosensor for ATP and DNA detection. Furthermore, we built a simple logic circuit based on this SDR mode with ATP and DNA strands as input. Incorporating an aptamer sequence into the DNA logic system and applying small molecules as input to tune the output signal of the logic circuit are very significant for the application of the DNA computing system in a wide range of fields. Realizing a disease marker sensitive smart drug delivery system will be quite promising if some relationship between the final output signal and drug release is established. This work was supported by the National Natural Science Foundation of China (Grants 21075116 and 21190040) and 973 projects (2010CB933600 and 2011CB911000).
Notes and references
Fig. 3 (a) Schematic diagram of the logic circuit with ATP and DNA strands as inputs. (b) Normalized FI at 630 nm of the different input modes of the circuit. FI at 630 nm of curve a in Fig. 1b is set as 1. Threshold value is set at 0.4 to judge the positive and negative output signals. The three binary numbers represent the input status of ATP, cABA and R, respectively. The data were collected from three independent experiments.
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