pH-Programmable DNA Logic Arrays Powered by Modular DNAzyme

Letter pubs.acs.org/NanoLett

pH-Programmable DNA Logic Arrays Powered by Modular DNAzyme Libraries Johann Elbaz,†,§ Fuan Wang,†,§ Francoise Remacle,‡ and Itamar Willner*,† †

The Institute of Chemistry, The Hebrew University of Jerusalem, Jerusalem 91904, Israel Chemistry Department, B6c, University of Liège, 4000 Liège, Belgium

‡

S Supporting Information *

ABSTRACT: Nature performs complex information processing circuits, such the programmed transformations of versatile stem cells into targeted functional cells. Man-made molecular circuits are, however, unable to mimic such sophisticated biomachineries. To reach these goals, it is essential to construct programmable modular components that can be triggered by environmental stimuli to perform different logic circuits. We report on the unprecedented design of artificial pH-programmable DNA logic arrays, constructed by modular libraries of Mg2+- and UO22+-dependent DNAzyme subunits and their substrates. By the appropriate modular design of the DNA computation units, pH-programmable logic arrays of various complexities are realized, and the arrays can be erased, reused, and/or reprogrammed. Such systems may be implemented in the near future for nanomedical applications by pH-controlled regulation of cellular functions or may be used to control biotransformations stimulated by bacteria. KEYWORDS: DNA, DNAzyme, logic gates, biocomputing, field-programmable, pH

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versatile stem cells are programmed to generate target functional cells, we report here on a modular approach to construct libraries of catalytic nucleic acids (DNAzymes) and their substrates. DNAzymes are of growing interest in the fields of DNA nanotechnology,32 bioanalysis, 33 and targeted therapy.34 We use pH as a field-programmable environmental stimulus that selects specific combinational circuits consisting of a complete set of logic gates. Furthermore, by controlling the pH of the systems, the transition between programs is achieved, and by the application of anti-input(s) strand(s), any of the input(s) value can be erased and reset to a new value. As light signals can be used to alter pH, we demonstrate the lightstimulated programming of the circuits, thus highlighting the possibility to interface such biocomputational circuits with digital electronics. Results and Discussions. The principles for the design of the pH-programmable devices are shown in Figure 1A. The system consists of a library of DNAzymes subunits (Box I) and their respective substrates (Box II). In the presence of the appropriate nucleic acid inputs, the guided assembly of the computation unit occurs (Box III). This unit consists of an

hemical circuits performing logic and computational operations hold great promise in future nanoengineering1,2 and nanomedicine.3,4 Such systems could be interfaced with nanoscale devices, where the logic circuits, responding to a trigger, may activate the device. Numerous logic gates based on functional molecular assemblies were reported,5−9 and the reconfiguration of logic gates by light as external trigger was discussed.10 Alternatively, the base-sequences in nucleic acids were used to encode instructive information to perform logic operation in solutions or surfaces.11−13 The DNA logic circuits may provide a versatile paradigm for autonomous nanomedicine, where the response of the system to a biomarker (input) leads to the logic formation of the therapeutic product (output).14 The incorporation of such logic circuits into existing biological environments could yield the in vivo control of biotransformations.15 DNA and other biomolecules have already been used as components of biocomputational circuits.16−23 Nucleic acid-based logic gate cascades14,24−26 or automata performing parallel logic operations have been reported.27,28 Also, logic control of gene expression29,30 and RNA systems that process intracellular information were realized.31 For complex information processing, it is, however, advantageous to be able to program the device to the extent that an environmental trigger selects the particular combinational circuit of logic operations. Inspired by nature, where © 2012 American Chemical Society

Received: January 5, 2012 Revised: January 30, 2012 Published: February 1, 2012 6049

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reconfiguring the base sequences associated with the DNAzyme subunits, diversity and enhanced complexity of the selected programs is achieved. The inputs I1 and I2 include the complementary domains I and I′ and these domains are conjugated to the noncomplementary regions II and II′, respectively. Whenever I1 or I2 act each separately as input (I1̅ I2 or I1I2̅ ) the domains I and II or I′ and II′ bind the respective library components. Whenever I1 and I2 act simultaneously as inputs (I1I2), interhybridization of domains I and I′ is energetically favored, and only the attachment of the library subunits to domain II and II′ is possible. Figure 2A depicts the construction of three pH-driven arrays (AND, OR, XOR), using Library I and inputs (I1 and I2) as shown in Figure 1C. By programming the system at pH = 5.2 (P5.2), only the interhybridization of inputs I1 and I2, yields the active UO22+DNAzyme assembly giving rise to the cleavage of the substrate and to the generation of fluorescence, resulting in an AND gate, F1(5.2) = I1I2. The respective truth-table and fluorescence intensities of this gate are shown in Figure 2B (blue). At P7.2, the I1 and I2 select each the respective subunits to yield the active DNAzyme structures, while the interhybridization of the I1 and I2 selects the UO22+-DNAzyme subunits, that are inactive at this pH. This leads to the XOR gate, F1(7.2) = I1̅ I2 + I1I2̅ ; see truth table and the respective fluorescence intensities, Figure 2B, green. At P6.0, the two DNAzymes exhibit activity and, thus, in the presence of the inputs I1 and I2, the two active DNAzyme structures are formed, giving rise to the OR gate (F1(6.0) = I1̅ I2 + I1I2̅ + I1I2). The respective truth table and fluorescence results are shown in Figure 2B, red (for fluorescence spectra results see Figure S4, Supporting Information). The operation of the three gates was further confirmed by electrophoresis experiments, Figure 2C (for detailed discussion of the electrophoresis experiments, see S5, Supporting Information). By altering the pH of the system any of these arrays can be erased and reconfigured to drive a new logic gate; see Figures 2D and S6, Supporting Information. Furthermore, any of the inputs can be erased and reset to any other input value. This is achieved by the addition of the anti-input(s), T1 or T2 separately or together, according to the current input value of the device (for example, I1I2 is erased by the addition of T1T2). These antiinput(s) strand(s) are fully complementary to the inputs, leading to the dissociation of the DNAzyme subunits from the inputs. (For experimental results demonstrating the erase/reset of the input(s) values, see Figure S7, Supporting Information). Moreover, by increasing the diversity of the constituents of the libraries, the pH-programmed parallel activation of gates can be accomplished. Supporting Information Figure S8 shows the activation of AND; Half-Adder and XOR using library II, and Supporting Information Figure S9 depicts the programmed parallel activation of Half-Adder; YES(I1)-XOR; Half-Subtractor using library III. The paradigm of field-programmable logic gates was extended to construct field-programmable logic circuits, where the direction and the function are controlled by the pH, Figure 3. For this purpose, we modify the structure of the substrate of the different gates, Figure 3A. We implement caged substrates, where region I includes the ribonucleobasecontaining cleavage site, and the complementary domains for the respective DNAzyme subunits. Region II includes the sequence corresponding to the input for the subsequent gate (or in the presence of several substrates, with similar region I and different region II, the inputs for fan-out circuits, Figure 3B). The regions II and III of the substrate are hybridized with

Figure 1. (A) General design of the computing unit module using libraries of DNAzyme subunits and substrates. (For specific sequences of the two different DNAzyme libraries and substrate see S1, Supporting Information.) (B) Schematic presentation of the pHprogrammable logic arrays. (C) pH-programmed logic arrays of variable complexities executed by different libraries components using similar inputs. (For detailed description of the composition of the different libraries, see S3, Supporting Information.)

input module, the DNAzyme subunits catalytic core module, and an output module that yields the appropriate outputs (Box IV). In the present study, the pH is used to select the circuit executed by the machine, consisting of the Mg2+- and UO22+dependent DNAzymes/substrates libraries, and nucleic acids used as inputs. While the Mg2+-dependent DNAzyme35 exhibits highest activity at pH = 7.2, and is inactive at pH = 5.2, the UO22+-dependent DNAzyme36 is active at pH = 5.2, and inactive at pH = 7.2 (see Figure S2, Supporting Information). Since they exhibit partial activities at pH = 6.0, the two DNAzymes can act cooperatively to drive a third circuit. While previous studies have implemented the environment-insensitive strand-displacement principle to drive logic circuits,24−26 the sensitivity of DNAzymes to environmental triggers enables the guided selection of logic circuits. Thus, by the selection of the different subunits from the library, three different logic circuits can be realized, Figure 1B. In the subsequent discussion we implement the same inputs and different compositions of the DNAzyme subunits/ substrates libraries to drive different programs of variable complexities, Figure 1C. Since the input module is independent from the DNAzyme module and output module, by altering the composition of the substrates included in the library or by 6050

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Figure 2. (A) Assembly of library I upon interaction with the corresponding inputs (I1 and I2), executing pH-programmable AND; OR and XOR gates. Throughout the paper, domains X and X′ in the respective inputs and DNAzyme subunits or substrates represent complementary base pair regions. (B) Fluorescence intensities of the different gates at different pH in the form of bars (AND, blue; OR red; XOR, green) and the respective truth-table. (C) Nondenaturated gel electrophoresis results of the AND; OR; XOR gates, lanes (a) I1; (b) I2; (c−e) the libraries of the DNAzyme subunits; (f) the substrate; (g−j) by programming the system at pH = 5.2 in the presence of (g) no inputs, (h) I1, (i) I2, (j) both inputs; (k−n) by programming the system at pH = 6.0, where in lanes (k) no inputs, (l) I1, (m) I2, (n) both inputs; (o−r) by programming the system at pH = 7.2, where in lanes (o) no inputs, (p) I1, (q) I2, (r) both inputs. (D) Time-dependent fluorescence changes upon programming/reprogramming the system upon altering the pH of the device. Arrows on top indicate the time of application of the different pH-programmed, where the fluorescence of the system is reset to zero upon the application of any new programs.

a “helper” nucleic acid that blocks the secondary input from random activation of the circuit. The hybridization of region III with the “helper” nucleic acid stabilizes the caged structure of the substrate but ensures the release of the input upon cleavage of the substrate. Figure 4A depicts the programming of three different circuits using the same library of components. At P5.2, the first-layer of the circuit is an AND gate, where only the UO22+-dependent DNAzyme is activated in the presence of both inputs (I1I2). The cleavage of the two substrates leads to the release of two strands (ST and VR) that act each as one input for the second-layer, whereas I3 is the second input for this layer that yields an InhibAND as second-layer gate. The output of the circuit F2(5.2) = I1I2I3̅ , and the fluorescence intensities of the cascade are depicted in Figure 4B. At P7.2, the first-layer activates a NOR gate. In the absence of inputs (I1̅ I2̅ ), the “activator” constituent (YNMW) assembles and activates the Mg2+-DNAzyme subunits, whereas any other input(s) values, prohibits the formation of any DNAzyme. Cleavage of the caged substrates leads to the release of input ST that together with I3 yield the AND gate as second-layer of the

Figure 3. (A) Design of the DNAzymes caged substrate consisting of a protected sequence, being cleaved by the DNAzymes in response to an input. (B) (i) Activation of a serial gate cascade using substrates with a variable cleavage domain (region I) and variable protected sequences (region II). (ii) The activation of a fan-out gate device uses a set of caged substrates consisting of a common cleavage domain (region I) and variable protected sequences (region II). 6051

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Figure 4. (A) Nucleic acid library consisting of the Mg2+- and UO22+-dependent DNAzyme subunits, their respective substrates and inputs I1, I2 and I3. The system activates three programs of logic circuits, through the substrate-metabolism mechanism and environmental conditions. (B) Fluorescence intensities of the different pH-programmed circuits in form of bars (F1- violet and F2- brown) and the respective truth table. (C,D) Time-dependent fluorescence changes upon the activation of the pH-programmable circuits shown in (A). The different programs are followed by the fluorescence intensities of the two fluorophores, F1 and F2. (C) Depicts the time-dependent fluorescence changes of F1 at the different programs. (D) Shows the time-dependent changes of F2 at the different pH.

circuit. The output of this gate cascade (F1(7.2) = I1̅ I2̅ I3), and the respective fluorescence intensities are shown in Figure 4B. Programming the system to P6.0 activates a third circuit. The NXOR gate is activated in the first-layer, since the cleavage of

the caged substrates proceeds in the absence of the inputs (I1̅ I2̅ ) or only in the presence of both inputs (I1I2). The cleavage of the caged substrates yields as before, the ST and VR strands as a fan out outputs of the first-layer and these together with I3 6052

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system for 8 min generated a solution at pH = 6.8, where the Mg2+-DNAzyme was activated, and the UO22+-DNAzyme was inhibited. The illumination of the system for a time-interval of 3 min generated a solution at pH = 6.2, where both DNAzymes were active. Nonetheless, it should be noted that the maximum pH change observed upon irradiation of MG corresponded to ΔpH = 1.2. We find that in order to run the three pH-driven programs within the relative pH changes one might change the relative concentrations of the two ions acting as the DNAzyme cofactors (Mg2+ and UO22+), Supporting Information Figure S13. Thus, while the different pH values dictate the computing circuits, one may further control the programs with different range of pH by means of the ratio Mg2+:UO22+. Such cofactordependent control over the pH programs suggests that the computing circuits may be adapted, also, for systems that provide limited pH changes (For further discussions see Supporting Information Figure S13). To conclude, the present study has introduced a new modular approach to construct pH-programmable logic arrays by the application of libraries of DNAzyme subunits and their substrates as functional constituents. By the modular design of the DNAzyme subunits and their substrates, combinational circuits of different complexities were realized, and the reusability and reprogramming features of the systems were demonstrated. The advantages of the field-programmable DNAzyme subunits-based logic arrays paradigm include the following: (i) The systems are enzyme-free and exhibit scalability. (ii) By the incorporation of other metal-iondependent DNAzymes, for example, operating at basic pHvalues, further diversity in the programming functions may be achieved. (iii) The possibility to switch the pH by light or electrical signals37 suggests that the programmed DNA circuits may be interfaced with digital electronics. (iv) Many cellular disorders are accompanied by intracellular pH changes. Thus, appropriate logic circuits may be programmed to perform dictated intracellular functions, such as cleavage of m-RNA33 or the release of an inhibiting aptamer.14 Although the present system is not optimized for any specific intracellular process and does not consider the possible toxicity of UO22+, our system highlights a future nanomedical applications of such logic circuits. Finally, inspired by nature, where cells perform complex information processing circuits, we demonstrated that a predesigned “tool-box” of DNA constituents can be programmed to implement different functions by environmental pH triggers.

generate the second-layer of the circuit, activating the two parallel gates (InhibAND and AND gates). The outputs of this circuit F1(6.0) = I1̅ I2̅ I3 + I1I2I3 and F2(6.0) = I1̅ I2̅ I3̅ + I1I2I3̅ and the respective fluorescence intensities are given in Figure 4B. The kinetics of the different circuits are provided in panels C and D. Furthermore, the performance of the two layer circuits was supported by electrophoresis results, where the products of the first-layer and of the second-layer gates were characterized (for a detailed description of the electrophoresis results see Supporting Information S10, S11). It should be noted that the circuits presented in this study represent examples of different achievable circuits using this paradigm (for a comprehensive description of the general field-programmable circuits see Supporting Information S12). We have further demonstrated that the programmed gates can be controlled by external light stimuli in order to highlight the possibility to interface such biocomputational circuits with digital electronics. Toward this end, we made use of the photoactive Malachite Green (MG), Figure 5. The photo-

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ASSOCIATED CONTENT

S Supporting Information *

Materials and methods, DNA sequences, and additional experiments. This material is available free of charge via the Internet at http://pubs.acs.org.

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Figure 5. Programmed gates controlled by external light stimuli, upon illumination of the photoactive Malachite Green (MG).

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected].

dissociation of MG controls the pH of the medium by the time interval of illumination. While in the absence of light the solution at pH = 5.6 activated the UO22+-DNAzyme, and the Mg2+-DNAzyme was almost fully inhibited, irradiation of the

Author Contributions §

Equal contribution to this study.

Notes

The authors declare no competing financial interest. 6053

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(35) Breaker, R. R.; Joyce, G. F. Chem. Biol. 1995, 2, 655−660. (36) Liu, J.; Brown, A. K.; Meng, X. L.; Cropek, D. M.; Istok, J. D.; Watson, D. B.; Lu, Y. Proc. Natl. Acad. Sci. U.S.A 2007, 104, 2056− 2061. (37) Frasconi, M.; Tel-Vered, R.; Elbaz, J.; Willner, I. J. Am. Chem. Soc. 2010, 132, 2029−2036.

ACKNOWLEDGMENTS Parts of the study were supported by the EU MOLOC project and by the Office of Naval Research, U.S.A. F.R. is Director of Research at Fonds National de la Recherche Scientifique (FNRS), Belgium. J.E. acknowledges a Converging Technologies Fellowship (Israel Science Foundation). We thank Professor R.D. Levine for the fruitful discussions.

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