EnzymeFree Unlabeled DNA Logic Circuits Based on

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Enzyme-Free Unlabeled DNA Logic Circuits Based on Toehold-Mediated Strand Displacement and Split G-Quadruplex Enhanced Fluorescence Jinbo Zhu, Libing Zhang, Tao Li, Shaojun Dong, and Erkang Wang*

As a basic material, silicon has played the role of overlord in the information technology (IT) materials field for several decades. Suffering from the gap between its finite capability and the infinite demand in development, scientists are searching for a more-advanced and powerful material. DNA is a promising engineering material in this field for its outstanding datastorage capacity and flexibility in design.[1] Recently, various DNA logic gates,[2] circuits,[3] even neural networks[4] and tiny circuit boards[5] have been reported, which prove its potential in computing science. However, the requirement for DNA nucleases or expensive chemical labels impedes the development of DNA computing and its application in a broader field. Thus, the establishment of enzyme-free and unlabeled DNA logic circuits as well as its potential application in disease diagnosis and therapy will be attractive and meritorious. A DNA strand displacement reaction mediated by toehold, a short single strand region at one end of a double helix strand, is an ideal approach to receive the input DNA signal and release the other strand as the out signal, which imitates the function of some DNA nucleases to output the short segments. Based on this reaction, the signal transmission can be performed among different gates without a special enzyme.[6] The fluorescent label is another reason for the high cost. Herein, we choose protoporphyrin IX (PPIX) as a signal reporter, because it can specifically bind to the split G-quadruplex whose formation can be controlled through hybridization induced by input DNA.[7] Utilizing the strand displacement reaction initiated by toehold and fluorescence of PPIX enhanced by split G-quadruplex (3:1), we constructed a series of DNA logic gates. Additionally, the input and output of these gates are all single-strand DNA (ssDNA) except the last fluorescent readout signal, which enables them to be easily cascaded into circuits and achieve more-complex functions. The final readout YES gate can be reset through strand

J. Zhu, L. Zhang, Dr. T. Li, Prof. S. Dong, Prof. E. Wang State Key Laboratory of Electroanalytical Chemistry Changchun Institute of Applied Chemistry Chinese Academy of Sciences Changchun, Jilin, 130022, P. R. China E-mail: [email protected] J. Zhu, L. Zhang, Dr. T. Li, S. Dong, Prof. E. Wang University of Chinese Academy of Sciences Beijing, 100039, P. R. China

DOI: 10.1002/adma.201205360

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displacement reaction. Furthermore, research about the transport and controlled release of drugs based on logic gates and circuits has arisen widespread interest in recent years.[8] As an important molecule in photodynamic diagnosis and therapy (PDT), PPIX can work as photosensitizer to absorb light and induce the photodynamic killing of tumor cells.[9] The binding between PPIX and G-quadruplex is largely dependent on the integrity of G-quadruplex structure.[7a,c,10] In this work, setting PPIX as a drug model, through modulating the formation of split G-quadruplex by input DNA, we performed a study of controlling the coalescence and release of PPIX in vitro. Combined with enzyme-free unlabeled DNA logic circuits, the drug release model is able to be regulated by several different signal sources, which demonstrates the potential application of this DNA computing system in drug delivery. Firstly, we constructed the basic single input YES and NOT logic gates. The two G-rich segments in solution bound to PPIX weakly. As shown in Figure 1a, when strand S was input, the split G-quadruplex (3:1) was formed since G1 and G2 got close to each other through hybridization with this input strand. The fluorescence of PPIX was dramatically enhanced after its binding to the split G-quadruplex (see Supporting Information, Figure S1a). It could work as a YES gate with an input of the strand S and output of the fluorescent signal. We defined the normalized fluorescent intensity above the threshold of 0.4 as “1” in the logic gate. A short six bases single strand at the 3’ end of strand S (the grey domain in the schemes) was reserved as toehold to interact with input strand I, which could hybridize with S and disband the split G-quadruplex (Figure 1b). Consequently, the fluorescence intensity of PPIX decreased dramatically to the original level (Supporting Information, Figure S1a,b). This is a typical phenomenon of a NOT gate. The YES gate is an ideal fluorescent readout for the input strand signal from the upper gates. Replacing the strand from different ends, we set up an OR logic gate to control the release of S and linked it with the YES gate. As shown in Figure 1c, strands C1 and C2 can displace S from different toeholds at the two sides of strand I (the green domains in the schemes) to give off S, which worked as an input of the YES gate to guide the formation of G-quadruplex and enhance the fluorescence of PPIX. The fluorescent results shown in Figure S1c,d in the Supporting Information testified that inputting either one of C1 and C2 is enough to release S. It is worth mentioning that strand I hybridized with S in a loop-stem form is to avoid the repeat of sequence between S and C1 or C2. The truth table is shown in Figure S2 in the Supporting Information.

© 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

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Figure 1. Schemes of logic gates: a) YES, b) NOT, and c) OR.

The AND logic gate is a very important gate. To achieve it we used a longer strand A1 to bind S (Figure 2a). A2 bound the remainder single part of A1 and blocked the toehold, which was designed for strand cA1 to displace S. Thus, cA1 hardly hybridized with A1 when A2 occupied the toehold. Another input strand cA2 could remove A2 from A1 for it is complementary to A2. After the addition of cA1 and cA2, A2 and A1 would be removed in succession and give off S to induce the high fluorescence intensity (Figure 2b,c). YES gate was used to report the output signal again. The circuit and truth table were shown in Figure 2d. Furthermore, native polyacrylamide gel electrophoresis (PAGE) has been used to identify this strand displacement mechanism. In Figure 2e, the DNA strands of corresponding bands have been figured out. Comparing lane 7 with 6, we found that after the addition of cA1 the band of S+A1 disappeared and two new bands emerged. Through comparing them with the band in lane 1 and 5, we drew the conclusion that cA1 replaced S on A1, so the band of the single strand S at the forefront of the gel could be observed. The consistency of material for input and output make these gates easily cascaded. Modulating the sequence of AND to control the release of C2, the input DNA of the above mentioned OR gate, we successfully combined these three gates into circuit to tune the fluorescence of PPIX. In Figure 3a, P1 bound C2 and P2 hybridized with the sticky end of P1 to block the toehold. After the addition of their complementary strands cP2 and cP1, P2 and P1 would be successively removed and C2 was released. As the input strand of OR gate, C2 opened the loop-stem structure of OR gate through the toehold on strand I and gave off S to induce the final fluorescent output signal (Figure S3). Certainly, for the property of OR gate, no matter whether cP1 or cP2 was input, S would be gave off as soon as C1 was present. The circuit and truth table were shown in Figure 3b,c. Furthermore, based on the above design we constructed another two logic gates, INHIBIT and IMPLICATION, by selecting different

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strands as input and output. The scheme, fluorescent results and truth table were shown in Figure S2, S4. Since PPIX plays an important role in PDT and possesses unique fluorescent property, we chose it as the drug model and investigated its controlled release through our DNA circuits in vitro. The trapping and release of PPIX can be modulated by the formation and dissociation of G-quadruplex. Here we integrated AND, OR, NOT and YES gates into an advanced circuit, where the release of PPIX was logically controlled by several different stimuli (Figure 4a). In the NOT gate, strand I assumed the responsibility of hybridizing with S to dissociate the G-quadruplex and set free PPIX. Taking the strategy of AND gate, we used N1 and N2 to bind I and block the toehold, respectively. Therefore, only the two input of AND, cN1 and cN2, were both present, would strand I be output into the NOT gate to disassemble the G-quadruplex and lead the release of PPIX. This assumption was confirmed by the conversion of the fluorescence intensity caused by the input (Figure 4b,c). The circuit and truth table were shown in Figure 4d and 4e, respectively. From another perspective, the connection of AND and NOT equates the NAND gate. Note that the release of PPIX is reversible. The binary hybrid S+I is the basic molecular platform for OR gate, thereby addition of any one of C1 or C2 was enough to displace the S on I and promote the regeneration of G-quadruplex to capture PPIX (Figure 4a,b,c). In sum, the two input of AND controlled the release of PPIX and the input strands of OR inhibited this process. Obviously, if some relationship was built between the input sequence and disease, or aptamer sequence was assembled into the circuit to sense special biomolecule, the release of drug would be able to be controlled by these meaningful factors. From this point of view, this system would be very useful in clinical diagnosis and therapy. Regeneration is another arduous challenge in the molecular computing field.[11] In our design, the final readout gate can be easily reset as shown in Figure 5a. Strand I hybridized with S



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Figure 2. a) Principle of the AND-YES circuit. b,c) Fluorescence emission spectra (b) and normalized fluorescence intensity at 630 nm (c) of the four input modes of AND gate: (1) no input, (2) cA1, (3) cA2, (4) cA1 and cA2. d) Logic circuit and truth table of AND-YES circuit. e) Native 15% polyacrylamide gel analysis of the AND gate. Different DNA samples were added into lanes 1–7. Lane 1: S; lane 2: S+A1; lane 3: S+A1+A2; lane 4: cA2+A2; lane 5: cA1+A1; lane 6: S+A1+A2+cA2; lane 7: S+A1+A2+cA2+cA1. The concentrations for each DNA in PAGE are all 2 μM.

Figure 3. a) Principle of AND-OR-YES circuit. b,c) Logic circuit (b) and truth table (c) of AND-OR-YES circuit.

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COMMUNICATION Figure 4. a) Principle of AND-NOT circuit. b,c) Fluorescence emission spectra (b) and normalized fluorescence intensity at 630 nm (c) of the different input modes of the circuit: (1) no input, (2) cN1, (3) cN2, (4) cN1 and cN2, (5) cN1, cN2 and C2. d,e) Logic circuit (d) and truth table (e) of AND-NOT circuit.

through the toehold to form state 1 and dissociated G-quadruplex. Then C1 took the similar approach to release S, which regenerated the split G-quadruplex and made the cycle back to state 2. The transformation between state 1 and 2 of YES gate was driven by input strand I and C1, which worked as fuel of this cycle and produced the waste of double strand I+C1. Native PAGE was employed to identify this mechanism, whose result was shown in Figure 5b. The band of S was present in lane 6 and 8 after the

addition of C1 in every cycle. The fluorescent data was shown in Figure S5. We can see that it could circulate for several times. To sum up, a series of unlabeled fluorescent logic gates (YES, NOT, OR, AND, INHIBIT, IMPLICATION and NAND) has been constructed in this work based on the split G-quadruplex enhanced fluorescence of PPIX. Adopting the toehold-mediated strand displacement reaction, the DNA output signal can be transmitted among different gates and these gates can be cas-

Figure 5. a) Scheme of the cycle transformation between different states of YES gate driven by I and C1. b) Native 15% polyacrylamide gel analysis of the cycle between states 1 and 2. Different DNA samples were added into lanes 1-8. Lane 1: 2 μM S; lane 2: 2 μM I; lane 3: 2 μM C1; lane 4: 2 μM I+C1; lane 5: 2 μM S+I; lane 6: 2 μM S+I+C1; lane 7: add another 2 μM I into 2 μM S+I+C1; lane 8: add another 2μM C1 compared with lane 7. Adv. Mater. 2013, 25, 2440–2444



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caded into circuit without enzyme. The photosensitive molecule PPIX is not only as a fluorescent probe but also a drug model investigated by us in this work. Controlled release of PPIX was realized by the AND-NOT circuit and it could be reset by the OR-YES circuit. In principle, any DNA sequence including specific gene fractions for some diseases can be used as the input for our built logic circuit,[3c,8c] which offers this system exciting prospects. From the perspective of application in the future, this enzyme-free unlabeled DNA logic circuit will be a competitive choice in DNA computing and drug delivery for its low cost and powerful functions.

Experimental Section Preparation of DNA Circuits: Sequences of DNA strands are listed in Table S1 in Supporting Information. The oligonucleotides were dissolved in water as stock solution and diluted with TEK buffer (10 mM Tris-HCl, 1 mM ethylenediaminetetra-acetic acid (EDTA), 0.2 M NaCl, 0.1 M KCl, pH 8.0) for hybridization in the logic circuit. In logic operation, the solution of basic logic DNA diluted with TEK buffer was first heated at 88 °C for 10 min, then slowly cooled down to room temperature (RT). Input DNA was added in step and incubated under RT for 30 min for each addition. G1, G2 and PPIX diluted with TEK buffer were added at last and incubated for 1 h before fluorescent test. The fluorescent analysis was performed in the TEK buffer with a final concentration of 0.6 μM for PPIX, 0.2 μM for G1, G2, S and 0.32 μM for other DNA strands. More experimental details can be found in the Supporting Information.

Supporting Information Supporting Information is available from the Wiley Online Library or from the author.

Acknowledgements The authors thank the support of the National Natural Science Foundation of China (Grants 21075116 and 21190040) and 973 projects (2010CB933600 and 2011CB911002). Received: December 31, 2012 Published online: February 28, 2013

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