DNA computing circuits using libraries of DNAzyme ...

LETTERS PUBLISHED ONLINE: 30 MAY 2010 | DOI: 10.1038/NNANO.2010.88

DNA computing circuits using libraries of DNAzyme subunits Johann Elbaz1, Oleg Lioubashevski1, Fuan Wang1, Franc¸oise Remacle2, Raphael D. Levine1 and Itamar Willner1 * Biological systems that are capable of performing computational operations1–3 could be of use in bioengineering and nanomedicine4,5, and DNA and other biomolecules have already been used as active components in biocomputational circuits6–13. There have also been demonstrations of DNA/RNA-enzyme-based automatons12, logic control of gene expression14, and RNA systems for processing of intracellular information15,16. However, for biocomputational circuits to be useful for applications it will be necessary to develop a library of computing elements, to demonstrate the modular coupling of these elements, and to demonstrate that this approach is scalable. Here, we report the construction of a DNA-based computational platform that uses a library of catalytic nucleic acids (DNAzymes)10, and their substrates, for the input-guided dynamic assembly of a universal set of logic gates and a half-adder/half-subtractor system. We demonstrate multilayered gate cascades, fan-out gates and parallel logic gate operations. In response to input markers, the system can regulate the controlled expression of anti-sense molecules, or aptamers, that act as inhibitors for enzymes. Our biocomputing approach is based on two libraries of nucleic acids, one consisting of subunits of DNAzymes and the second their substrates (Fig. 1a, boxes I and II). In the presence of the appropriate nucleic acid inputs, the simultaneous selection of pre-designed DNAzyme subunits and substrates from these respective libraries results in the assembly of the computational unit (box III) in two modules. The ‘input module’, which controls the gate functionality, consists of the input strands and ‘recognition arms’ of the DNAzyme subunits, and the ‘processing module’ includes the catalytic DNAzyme core that binds to the substrates. The input-guided assembly of the gate unit results in the cleavage of the substrate and releases the product strand. Thus, the independent structures of the input and processing modules provide diversity and modularity in the computational elements. In earlier DNA computing systems1,9, the inputs triggered transformation of the pre-designed ‘caged’ nucleic acid structure into active configurations of the specific gate functionality. Also, in most of those systems1,7–9, the structures operated as single-use gates. In contrast, we introduce a new gate adaptation principle in which the inputs guide the assembly and functionality of the gates through the selection of the corresponding subunits. The versatility of our method lies in the ability to select individual subunits to assemble different gates, and also in the fact that the gates are non-destructible. DNAzymes17,18 are of growing interest in the fields of DNA nanotechnology19, bio-analysis20,21 and targeted therapy22,23.The Mg2þ-dependent E6-type DNAzyme24 catalyses hydrolytic cleavage of a ribonucleobase (rA)-containing DNA substrate with a catalytic rate of 0.01 min21 (Fig. 1b). The use of DNAzyme subunits to

construct the computing elements is shown and explained in Fig. 1c,d. The construction of the XOR gate is outlined in Fig. 2a. The system consists of the DNAzyme subunits (1)–(4), the fluorophore/quencher-labelled substrate (5), and the inputs I1 and I2 (6) and (7). In the presence of I1 or I2, two different DNAzyme structures are formed, leading to the cleavage of the mutual substrate and to the generation of fluorescence (‘True’ output). Triggering the system with both inputs, I1 and I2, results in the formation of the energetically favoured duplex between (6) and (7) (DG8 ≈ 260 kcal mol21, compared to DG8 ≈ 214 kcal mol21 for the duplex structures between the inputs and the DNAzyme subunits, where DG8 correspond to the free energy changes upon the formation of the respective duplexes). This prohibits the formation of the active DNAzyme and the generation of fluorescence signals (‘False’ output). The experimental results of this system are presented in Fig. 2b, showing that fluorescence is triggered only upon activation by I1 or I2 alone, and is extinguished in the presence of I1 and I2, consistent with the operation of a XOR gate (for kinetic results, see Supplementary Fig. S1). Using an analogous approach, AND, InhibAND, NAND and NOR gates were constructed (Supplementary Figs S2–S5). The approach was then extended to activate half-adder (HA) and half-subtractor (HS) systems in a single test tube (Fig. 2c). An HA device produces Sum and Carry outputs, whereas an HS produces Difference and Borrow outputs. Parallel activation of the HA and HS devices requires the implementation of the AND, XOR and InhibAND gates in a single test tube. This was demonstrated by applying the same two inputs (I3 and I4) in a system with a library of DNAzyme subunits (1)–(4) and (8)–(10) and a collection of substrates (12), (5) and (11), each labelled with a different fluorophore (F1 , F2 , F3). Inputs I3 and I4 select the respective DNAzyme subunits from the library. Each of the fluorophores generates the output of a single gate (F1 ¼ AND; F2 ¼ XOR; F3 ¼ InhibAND). The fluorescence intensities corresponding to the output of the three gates are presented in Fig. 2d. The Carry bit output (AND gate) is probed by F1 , l ¼ 610 nm, and is observed only upon interaction with both inputs (I3, I4). The Sum and Difference bits (given by the same XOR gate) are presented by F2 , l ¼ 520 nm, which gives a True output in the presence of either I3 or I4, but a False output upon triggering by both inputs, due to preferred interinput hybridization. The Borrow bit output (InhibAND gate), given by F3 , l ¼ 710 nm, corresponds to a True value in the presence of I4 only. The fluorescence values resulting from the parallel operation of the HA and HS computation modules and the corresponding truth tables are presented in Fig. 2e. (For electrophoresis experiments supporting the AND gate, see Supplementary Fig. S6.) Challenges in DNA computing include achieving communication between different devices and scalability of the device

1

The Institute of Chemistry, The Hebrew University of Jerusalem, Jerusalem 91904, Israel, 2 Chemistry Department, B6c, University of Lie`ge, 4000 Lie`ge, Belgium. * e-mail: [email protected]

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DOI: 10.1038/NNANO.2010.88

Box I Box III Input module

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C A A T

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Input

Q

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Figure 1 | Design of the DNA computing module. a, General design of a computing unit module using libraries of DNAzyme subunits and substrates. b, The Mg2þ-dependent E6-type DNAzyme/substrate structure. c, E6-type DNAzyme divided into two subunits (I and II) that cannot assemble to form an active DNAzyme due to the limited six base pair complementarity of the subunits and the substrate. The DNAzyme subunits are tethered at their 3′ and 5′ ends to variable sequences that serve as recognition arms and provide the diversity of the subunits library (green). The fluorophore-tagged substrates of the DNAzymes (III) include the conserved sequence TrAGG tethered at the 3′ and 5′ ends to variable base sequences (yellow), leading to the library of substrates. d, Input-guided assembly of the DNAzyme and schematic cleavage of the fluorophore-tagged substrate. In the presence of the appropriate inputs (IV) complementary to the ‘recognition arms’ of the DNAzyme subunits, and in the presence of the appropriate substrate, the catalytic DNAzymes are synergetically stabilized by the input/substrate components, leading to cleavage of the substrate and separation of the subunits. The labelling of the substrates with appropriate fluorophore-quencher units leads, upon cleavage, to a fluorescence output, signalling the respective gate activity.

modules, and elimination of leakage and crosstalk. To this end, we have designed a caged substrate in which the output strand is protected until its release by the input-assembled DNAzyme. DNAzyme-stimulated cleavage of the substrates yields nucleic acid outputs that act as inputs for activating gates cascades or fan-out gates, thus providing multilayer circuits. Figure 3a depicts the configuration of the caged substrate, which consists of a ribonucleobasecontaining sequence (17) that is partially hybridized with the nucleic acid (18) in regions II and III. Region I contains the DNAzyme cleavage site TrAGG, and the complementary domains acting as the substrate for the DNAzyme. Region II includes base sequences C and D, which provide the input for the subsequent gate. The protected sequence C–D is inactive before cleavage of the substrate, because the duplex (17)/(18) is energetically favoured (DG ≈ 240 kcal mol21) compared to the duplex of C–D with the DNAzyme subunits of the next layer (DG ≈ 214 kcal mol21). Note that duplex III in (17)/(18) is longer than duplex II (22 base pairs, compared to 10 base pairs) and duplex III, therefore, provides stabilization for blocking domain C–D in the caged substrate. In the presence of the appropriate input and DNAzyme subunits, the caged substrate is cleaved in region I, synergetic stabilization of 418

the two duplex domains (regions II and III) is eliminated, and the protected sequence C–D is released. The single-stranded nucleic acid (C–D) then acts as an input for the subsequent gate. Although for the gate cascade the cleavage domains and the protected output sequences are different (Fig. 3b), the caged substrates for the fan-out gates include identical cleavage domains, but different protected output sequences (Fig. 3c). Figure 3d presents a cascade of YES2AND2InhibAND gates using a library of DNAzyme subunits (3), (4) and (19)–(22), the caged substrates as duplexes (15)/(16) and (17)/(18), the fluorophore-quencher-functionalized substrate (5), and inputs I5 (23), I6 (24) and I7 (25). In the presence of input I5 (23), the appropriate DNAzyme subunits (21) and (22) are selected from the library and assemble the YES gate with caged substrate (15)/(16). The cleaved substrate yields the strand R1EF, which acts as input for the AND gate. Although strand R1EF alone cannot form any active DNAzyme with the library components, in the presence of the second input I6 (24), the DNAzyme structure corresponding to the AND gate is formed. This results in the cleavage of substrate (17)/(18) and the release of strand R2CD, which, together with the auxiliary input I7 (25), act as inputs for the third-layer InhibAND gate. Although

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I (2) J (1) J (3) I (4) hν2

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I

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Figure 2 | Parallel activation of multigate systems. a, Organization of the DNAzyme subunits and the fluorophore-tagged substrate upon interaction with the corresponding inputs that activate the XOR gate. Throughout the paper, domains X and X′ in the respective inputs and DNAzyme subunits or substrates represent complementary base pair regions. b, Fluorescence intensities generated by the system in the presence of no input (i), input I1 (1,0) (ii), input I2 (0,1) (iii) and both inputs I1 and I2 (1,1) (iv). Difference in fluorescence intensities are also shown in the form of bars, together with a XOR gate truth table. c, Parallel activation of the three logic gates AND, XOR and InhibAND using the common inputs I3 and I4, the library of DNAzyme subunits and the fluorophore-tagged substrates (F1, F2 and F3 represent the fluorescence readout signals for the different gates, respectively). This system yields HA and HS devices. d, Fluorescence intensities resulting from the activation of the different gates: XOR gate, emission of F2 ¼ Fluorescein, lmax ¼ 520 nm; AND gate, emission of F1 ¼ ROX, lmax ¼ 610 nm; InhibAND gate, emission of F3 ¼ Cy5.5, lmax ¼ 710 nm. All the systems are activated by the common inputs I3 and I4: black (0,0) (i); blue (1,0) (ii); green (0,1) (iii); red (1,1) (iv). e, Difference fluorescence intensities (DF) of the different gates in the form of bars (XOR, blue; AND, red; InhibAND, yellow) and a truth table (DF corresponds to the difference between the fluorescence intensity generated by the gate and the fluorescence of the intact substrate before cleavage by the DNAzyme). f, Logic scheme for the HA and HS modules. NATURE NANOTECHNOLOGY | VOL 5 | JUNE 2010 | www.nature.com/naturenanotechnology

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DOI: 10.1038/NNANO.2010.88

Region I

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rA

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Figure 3 | Scalability of logic gates using gate cascades and fan-out. a, Design of the Mg2þ-dependent DNAzyme caged substrate consisting of a protected sequence, being cleaved by the DNAzyme in response to an active input. b, Activation of a serial gate cascade using substrates with a variable cleavage domain (region I) and variable protected sequences (region II). c, Activation of a fan-out gate device using a set of caged substrates with a common cleavage domain (region I) and variable protected sequences (region II). d, Nucleic acid library consisting of the Mg2þ-dependent DNAzyme subunits, their respective substrates and inputs I5, I6 and I7, which activate the serial gate cascade YES–AND–InhibAND through the substrate-metabolism mechanism. e, Change in fluorescence intensities (DF) of the YES–AND–InhibAND cascade outputs upon activation of the gate cascade by inputs I5, I6 and I7.

I7 (25) cannot generate any active DNAzyme, the released strand R2CD assembles the active DNAzyme, leading to the cleavage of (5) and resulting in fluorescence. In the presence of the two inputs, formation of duplex R2CD/I7 is favoured, and the formation 420

of any DNAzyme is prohibited. Note that fluorescence signal F2 is generated only if all of the components are present in the library, and only if all three cascaded gates are activated (see Fig. 3e for results). Control experiments revealed that in the absence of any

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I8 = F

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hν1

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M F1

R2−

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Figure 4 | Design of fan-out gates and the applicative of logic gates for nanomedicine. a, Nucleic acid library consisting of Mg2þ-dependent DNAzyme subunits, their respective substrates and input I8, which activate the fan-out YES gates. b, Fluorescence spectra corresponding to fan-out activation of YES gates 2 and 3 through primary activation of YES gate 1, which releases inputs R2CD and R3AB. (i) and (ii) represent fluorescence intensities of the respective gates in the absence or presence of input I8 (YES gate 2, emission lmax ¼ 510 nm; YES gate 3, emission lmax ¼ 610 nm). c, Fluorescence changes (DF) upon activation of gates 2 (blue) and 3 (red) by I8. d, Activation of the release of the anti-thrombin aptamers using a library of the Mg2þ-DNAzyme subunits and a caged aptamer substrate. e, Fluorescence spectra of the Rhodamine-110 fluorophore upon hydrolysis of (31) by thrombin: activation of the logic device by input I5 (i) and upon interaction of the thrombin with the logic device in the absence of I5 (ii).

of the library components, no cleavage of (5) (and formation of the fluorescent signal) occurred. Using a similar concept, the YES2YES2YES gate cascade was assembled (for results, see Supplementary Figs S7–S9). The DNAzyme-induced ‘metabolic’ cleavage of the substrate was then applied to fan-out the YES gates (Fig. 4a). The two substrates (17)/(18) and (26)/(27) include caged inputs R3AB and R2CD, respectively. Nucleic acids (3), (4), (10) and (20)–(22) are subunits for the self-assembly of

the different DNAzymes. The primary YES gate is activated by input I8 (EF), leading to the cleavage of the two substrates (17)/(18) and (26)/(27) and yielding the outputs R3AB and R2CD, which act as inputs for two parallel YES gates that activate the cleavage of substrates (12) and (5), respectively (Fig. 4b). The activation of serial or parallel gates through the metabolic cleavage of the caged substrates represents a major advance in biocomputing, because the DNAzyme-based cascaded gates demonstrate scalability

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of the circuits. One should realize, however, that the inputs generated by the gate cascades, as expected, weaken with increasing depth of the circuit. Furthermore, the non-stoichiometric generation of inputs and required inter-input hybridization (1,1 state) might hamper operation of the cascade. Nonetheless, the presented approach allows tailoring of an appropriate substrate for amplification of the gate output by a feedback mechanism (Supplementary Fig. S10). This path enables tuning of the content of the released output, relatively to the other gates, as well as amplification of the output. It should be noted that, by appropriately designing the inputs, all individual gates and all parallel gates can be constructed. In fact, for parallel gates, the inputs consist of a combination of sequence domains that correspond to the inputs of the individual gates. (For instructions regarding input design see the Supplementary Information, Discussion and Table S1.) The resulting constructs of logic gates translate input strands into pre-designed nucleic acid outputs. Thus, DNAzyme-based logic devices may contribute to the design of biotherapy methods, where input biomarkers are translated into anti-sense or protein inhibitors, released as outputs. This may be exemplified by a YES gate that yields the thrombin-binding aptamer (TBA) using a nucleic acid input, as a model for the biomarker. Thrombin is a hydrolytic enzyme participating in blood clotting, and high levels of thrombin are generated following brain haemorrhage or trauma, causing damage to brain cells or oedema25. The TBA consists of a G-rich 15-mer nucleic acid that self-assembles into a G-quadruplex that binds to thrombin and inhibits its activity26. The input-stimulated release of TBA is described in Fig. 4d. The DNAzyme subunits (1) and (2) and the caged substrate consisting of duplex (29)/(30), which includes the caged TBA sequence, form the components of the system. Upon activation of the system with input I5, the substrate is cleaved, and the released TBA inhibits the hydrolytic activity of the thrombin. This activity was probed by the cleavage of the Rhodamine 110-labelled argininecontaining peptide (31), which triggers the fluorescence of peptide de-protected fluorophore. Figure 4e shows that although the noninhibited thrombin effectively yields the fluorophore, the inputstimulated release of the aptamer inhibits the proteolytic activity of the thrombin, and only 40% of the enzyme activity is retained. Thus, the logic system demonstrates control of enzyme activity. In fact, any biomarker may activate a YES gate and be translated into an input sequence that activates the DNAzyme computation system. In conclusion, the present study has introduced a new proteinfree biocomputing platform based on a library of DNAzyme subunits, pre-designed substrates and instructive inputs. The uniqueness of the method lies in the modularity of the gate construct, the input-guided assembly of computing circuits, and the fact that computing elements are non-degradable following operation of the gates. We demonstrate the construction of a universal set of two-input logic gates leading to HA and HS computational modules in a single test tube. Furthermore, the study has demonstrated the modularity and scalability of the computing elements, by constructing multilayered logic circuits and executing gate multiplication with a fan-out scheme. As we have constructed a universal set of logic gates, any Boolean logic unit may be assembled by the appropriate combination of gates. Finally, we discussed the use of the DNAzyme-based computing elements for future nanomedicine applications. Received 9 October 2009; accepted 7 April 2010; published online 30 May 2010; corrected after print 9 February 2011

References 1. Stojanovic, M. N. Molecular computing with deoxyribozymes. Prog. Nucleic Acid Res. Mol. Biol. 82, 199–217 (2008). 422

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2. De Silva, A. P. & Uchiyama, S. Molecular logic and computing. Nature Nanotech. 2, 399–410 (2007). 3. Benenson, Y. Biocomputers: from test tubes to live cells. Mol. Biosyst. 5, 675–685 (2009). 4. Riehemann, K. et al. Nanomedicine—challenge and perspectives. Angew. Chem. Int. Ed. 48, 872–897 (2009). 5. Simmel, F. C. Towards biomedical applications for nucleic acid nanodevices. Nanomedicine 2, 817–839 (2007). 6. Mao, C. D., LaBean, T. H., Reif, J. H. & Seeman, N. C. Logical computation using algorithmic self-assembly of DNA triple-crossover molecules. Nature 407, 493–496 (2000). 7. Voelcker, N. H., Guckian, K. M., Saghatelian, A. & Ghadiri, M. R. Sequenceaddressable DNA logic. Small 4, 427–431 (2008). 8. Seelig, G., Soloveichik, D., Zhang, D. Y. & Winfree, E. Enzyme-free nucleic acid logic circuits. Science 314, 1585–1589 (2006). 9. Shlyahovsky, B., Li, Y., Lioubashevski, O., Elbaz, J. & Willner, I. Logic gates and antisense DNA machine operating on translator scaffold. ACS Nano 3, 1831–1843 (2009). 10. Stojanovic, M. N. & Stefanovic, D. A deoxyribozyme-based molecular automaton. Nature Biotechnol. 21, 1069–1074 (2003). 11. Penchovsky, R. & Breaker, R. R. Computational design and experimental validation of oligonucleotide-sensing allosteric ribozymes. Nature Biotechnol. 23, 1424–1433 (2005). 12. Benenson, Y., Paz-Elizur, T., Adar, R., Keinan, E. & Shapiro, E. Programmable and autonomous computing machine made of biomolecules. Nature 414, 430–434 (2001). 13. Niazov, T., Baron, R., Lioubashevski, O., Katz, E. & Willner, I. Concatenated logic gates using four coupled biocatalysts operating in series. Proc. Natl Acad. Sci. USA 103, 17160–17163 (2006). 14. Benenson, Y., Gil, B., Ben-Dor, U., Adar, R. & Shapiro, E. An autonomous molecular computer for logical control of gene expression. Nature 429, 423–429 (2004). 15. Rinaudo, K. et al. A universal RNAi-based logic evaluator that operates in mammalian cells. Nature Biotechnol. 25, 795–801 (2007). 16. Win, M. N. & Smolke, C. D. Higher-order cellular information processing with synthetic RNA devices. Science 322, 456–460 (2008). 17. Breaker, R. R. DNA enzymes. Nature Biotechnol. 15, 427–431 (1997). 18. Lu, Y. & Liu, J. Functional DNA nanotechnology: emerging applications of DNAzymes and aptamers. Curr. Opin. Biotechnol. 17, 580–588 (2006). 19. Willner, I., Shlyahovsky, B., Zayats, M. & Willner, B. DNAzymes for sensing, nanobiotechnology and logic gate applications. Chem. Soc. Rev. 37, 1153–1165 (2008). 20. Kolpashchikov, D. M. A binary deoxyribozyme for nucleic acid analysis. ChemBioChem 8, 2039–2042 (2007). 21. Elbaz, J., Moshe, M., Shlyahovsky, B. & Willner, I. Cooperative multicomponent self-assembly of nucleic acid structures for the activation of DNAzyme cascades: a paradigm for DNA sensors and aptasensors. Chem. Eur. J. 14, 3411–3418 (2009). 22. Khachigian, L. M. Catalytic DNAs as potential therapeutic agents and sequencespecific molecular tools to dissect biological function. J. Clin. Invest. 106, 1189–1195 (2000). 23. Basu, S., Sriram, B., Goila, R. & Banerjea, A. C. Targeted cleavage of HIV-1 coreceptor-CXCR-4 by RNA-cleaving DNA-enzyme: inhibition of coreceptor function. Antivir. Res. 46, 125–134 (2000). 24. Breaker, R. R. & Joyce, G. F. A DNA enzyme with Mg2þ-dependent RNA phosphoesterase activity. Chem. Biol. 2, 655–660 (1995). 25. Chapman, J. Thrombin in inflammatory brain diseases. Autoimmun. Rev. 5, 528–531 (2006). 26. Bock, L. C., Griffin, L. C., Latham, J. A., Vermaas, E. H. & Toole, J. J. Selection of single-stranded DNA molecules that bind and inhibit human thrombin. Nature 355, 564–566 (1992).

Acknowledgements Parts of this research are supported by the EC Project MOLOC and by the Office of Naval Research, USA. 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).

Author contributions J.E. designed the systems, performed the experiments, analysed the results and participated in the formulation of the paper. O.L. participated in designing the system, discussing the research results and the formulation of the paper. F.W. participated in designing the system and performed the experiments. R.D.L and F.R. participated in discussing the research results and the formulation of the paper. I.W. supervised the project, evaluated the research results and participated in the formulation of the paper.

Additional information The authors declare no competing financial interests. Supplementary information accompanies this paper at www.nature.com/naturenanotechnology. Reprints and permission information is available online at http://npg.nature.com/reprintsandpermissions/. Correspondence and requests for materials should be addressed to I.W. NATURE NANOTECHNOLOGY | VOL 5 | JUNE 2010 | www.nature.com/naturenanotechnology

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corrigendum

DNA computing circuits using libraries of DNAzyme subunits Johann Elbaz, Oleg Lioubashevski, Fuan Wang, Françoise Remacle, Raphael D. Levine and Itamar Willner Nature Nanotechnology 5, 417–422 (2010); published online: 30 May 2010; corrected after print: 9 February 2011. In the version of this Letter originally published, components of the systems illustrated in Figs 3a–d, 4a and 4d were incorrectly labelled. In the Supplementary Information, components of the systems illustrated in Figs S7a, S9a–c and S10 were also incorrectly labelled. These errors have now been corrected in the HTML and PDF versions of the text, and in the Supplementary Information.

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SUPPLEMENTARY INFORMATION doi: 10.1038/nnano.2010.88

Supplementary Information for the paper:

“DNA Computing Circuits Using Libraries of DNAzyme Subunits”

Johann Elbaza, Oleg lioubashevskia, Fuan Wanga, Françoise Remacleb, Raphael D. Levinea and Itamar Willner a*

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The kinetics of the XOR gate operation is depicted in Figure S1.

Figure S1: Time-dependent fluorescence changes of the XOR gate shown in Figure 2A in the paper: (a) no inputs, (b) input I1, (1,0), (c) input I2, (0,1), (d) the two inputs I1 and I2, (1,1).

2

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Figure S2 depicts the AND gate and its experimental results.

Figure S2: (A) Assembly of the DNAzyme construct for the AND gate. (B) Fluorescence intensities generated by the system in the presence of (a) no inputs, (b) input I9 (1,0), (c) input I10 (0, 1), (d) two inputs I9 and I10 (1, 1). Also, the same fluorescence intensities, in the presence of the different inputs, are presented in form of bars, and the AND gate truth table is presented.

The

system

consists

of

the

two

DNAzyme

subunits

(8)

and

(9),

the

fluorophore/quencher-labeled substrate (12), that its cleavage provides the readout for the gate operation, and the inputs I9 and I10, (32) and (33), respectively. The inputs I9 and I10 include partial complementarity to the subunits, yet each of these inputs alone cannot assemble the subunits into an active DNAzyme structure, and thus the "0" output is generated. In the presence of both inputs, I9 and I10, cross hybridization between domain H, H' of the inputs allows the selection of the two DNAzyme subunits and the synergistically-stabilized supramolecular DNAzyme structure that leads to the cleavage of the substrate (12), and the generation of the fluorescence, F1. nature nanotechnology | www.nature.com/naturenanotechnology

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Figure S3 depicts the InhibAND gate and its experimental results.

Figure S3: (A) Schematic organization of the DNAzyme subunits and its fluorophore-tagged substrate upon interaction with the respective inputs that activate the InhibAND gate. (B) Fluorescence intensities generated by the system in the presence of (a) no input (b) input I11 (1,0) (c) input I12 (0, 1) (d) two inputs I11 and I12 (1, 1). Also, the fluorescence intensities in the prescence of different inputs are presented in form of bars, and the InhibAND gate truth table is presented.

In the presence of I12 the DNAzyme structures are formed, leading to the cleavage of the substrate, leading to the generation of the fluorescence. Triggering the system with both inputs, I11 and I12, results in, however, the formation of the energetically favored duplex between (34) and (35) G ~ -60 kcal.mol-1, as compared to G ~ -14 kcal.mol-1 for the duplex structures between the inputs and the DNAzyme subunits.

This prohibits the

formation of DNAzyme structure and the generation of a fluorescence signals. Thus, a False output is observed upon triggering the system with the two inputs. Next, we use the DNAzyme assembly to build universal NAND and NOR gates. The operation of NAND and NOR gates is based on the co-addition to the library of DNAzyme 4

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subunits of the nucleic acids (23) and (38), respectively, that pre-organize the active DNAzyme structures.

Figure S4 depicts the configuration of NAND gate and the respective experimental results.

Figure S4: (A) Schematic organization of the DNAzyme subunits and its fluorophore-tagged substrate upon interaction with the respective inputs that activate the NAND gate. (B) Fluorescence intensities generated by the system in the presence of (a) no input (b) input I13 (1,0) (c) input I14 (0, 1) (d) two inputs I13 and I14 (1, 1). Also, the fluorescence intensities in the presence of different inputs are presented in form of bars, and the NAND gate truth table is presented. In the absence of any input the co-added nucleic acid (23) bridges the DNAzyme subunits, leading to an active DNAzyme structure that cleaves the substrate and yield a TRUE output. In the presence of either input I13, (36), or I14, (37), that include, respectively, domains A1' or B1' complementary to the co-added strand (23), the DNAzyme structure between the co-added strand (23) and DNAzyme subunits is energetically favored (complementarity of 12 bases vs 10 bases, for A'-A VS A1'-A and 13 bases vs 11 bases B'-B vs B1'-B, respectively). This leads to the cleavage of the substrate and yields a TRUE output. In the presence of both inputs, I13 and I14, the hybridization of the inputs to the co-added nature nanotechnology | www.nature.com/naturenanotechnology

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strand (23) results in the formation of the T-shaped duplex, induced by the complementarity of P and P', that eliminates the active DNAzyme structure, leading to the FALSE output (the duplex P/P' synergistically stabilizes the A1/A1' and B1/B1'duplexes). Figure S5 depicts the design of the NOR gate, and the respective experimental results.

Figure S5: (A) Schematic organization of the DNAzyme subunits and its fluorophore-tagged substrate upon interaction with the respective inputs that activate the NOR gate. (B) Fluorescence intensities generated by the system in the presence of (a) no input (b) input I15 (1,0) (c) input I16 (0, 1) (d) two inputs I15 and I16 (1, 1). Also, the fluorescence intensities in the presence of different inputs are presented in form of bars, and the NOR gate truth table is presented.

In the absence of any input the co-added strand (38) bridges the DNAzyme subunits, and an active DNAzyme structure cleaves the substrate, that yields a TRUE output. In the presence of either input I15, (39), or I16, (40), that include, respectively, domains A' or B', complementary to the co-added strand (38), or both inputs, the DNAzyme structure between the co-added strand (41) and DNAzyme subunits is eliminated by energetically favored 6

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duplexes between co-added strand and inputs (complementarity of 27 bases vs 12 bases, for AC-A'C', A-A' and 30 bases vs 13 bases for BD-B'D', B-B', respectively). This leads to the FALSE output of the gate.

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Figure S6 shows the gel electrophoresis results following the AND gate operation.

Figure S6: Acrylamide electrophoresis of the AND gate system: Control entries: (a) input I3, (b) input I4 (c) DNAzyme subunit (8) (d) nucleic acid substrate (15). Entries (e) to (f) correspond to different gate states: (e) no inputs, (f) input I3 only, (1,0) g) input I4 only, (0,1) (h) the two inputs I3 and I4, (1,1).

One may realize that only the activation of the system with the two inputs I3 and I4 leads to disappearance of the bands corresponding to I3 and I4 and appearance of the bands corresponding to the I3/I4 hybrid, and the output M' of the AND gate. These results are consistent with the AND gate operation.

8

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Figure S7 depicts the YES gates cascade and the respective experimental results.

Figure S7: (A) The nucleic acid library consisting of the Mg2+-dependent DNAzyme subunits, their respective substrates and the input I5 that activate the serial gate cascade YES-YES-YES by the substrate metabolism mechanism.(B) The fluorescence spectra of the YES-YES-YES cascade gates upon the activation with input I5 : (a) no input (b) in the presence of I5. (C) Fluorescence intensity of the YES-YES-YES gate cascade in form of bars presentation: (a) control, in the absence of any component of the library of the Mg2+-dependent DNAzyme subunits or their respective caged substrates, in the presence of I5; (b) in the presence of all the component of the library of the Mg2+-dependent DNAzyme subunits and their respective caged substrates without I5; (c) in the presence of all the component of the library of the Mg2+-dependent DNAzyme subunits, their respective caged substrates and I5. nature nanotechnology | www.nature.com/naturenanotechnology

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Figure S8 shows the kinetics of YES gates cascade.

Figure S7: Time-dependent fluorescence changes of the YES gates cascade: (a) single YES gate output activated by input; (a') single YES gate with no input; (b) two coupled YES gates activated by input; (b') two coupled YES gates with no input; (c) three wired YES gates activated with input; (c') three wired YES gates with no input.

One may realize that longer set-on time intervals are observed as the number of wired gates increases.

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Figure S9 shows the amplification feedback method:

Figure S9

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Amplification of the gate output signals in the layered circuits using feedback mechanism. The gate-generated outputs that act as inputs for subsequent layer are weakened with the depth of the circuit. This phenomenon is, however, natural for DNA computing schemes. Our computing paradigm that yields the layered circuits enables, however, to extend the design of the substrates to structures that lead to a feedback mechanism that amplifies and enriches the output signals for the subsequent gate, and enables the tuning of the appropriate stoichiometry of the inputs. One should note that in the layered circuit depicted in Figure 3 and Figure 4 the cleavage of the caged substrate led to a nucleic acid product acting as output for the next gate, and to a "waste" nucleic acid product that had no utility. One may, however, use the "waste" product as an activating unit of the feedback amplification as outlined in Figure 9S. The cleavage of the caged substrate shown in Figure 9S (A) leads to the output nucleic acid that acts as input for the next gate and to the nucleic acid strand ІІa that activates the feedback mechanism. The library of DNAzyme subunits includes now, as additional component, the hairpin structure (42) that is opened by the product strand N'D1. The resulting open hairpin includes, however, the subunit (S' - I) that in the presence of subunit (41) assembles into the active Mg2+-dependent DNAzyme that cleaves the fluorophore-quencher-functionalized substrate, (5), (or re-cleaves the substrate for the subsequent layer), Figure 9S (B). The amplified generation of the output nucleic acid by feedback mechanism is schematically depicted in Figure 9S (C). The library consists of the subunits (10), (20) and (3), (4) that provide the elements for the assembly of the “gate 1” and “gate 2”, respectively. The duplex (17)/( 18) represents the caged substrate for the layered circuit. The substrate (5) acts as the labeled substrate for the readout of the gate cascade output. The library of subunits includes, in addition, the hairpin structure (44) that upon interaction with the released strand ND’ leads to the assembly of the DNAzyme that re-cycles the formation of strand ND’. Figure 9S (D) shows that the hairpin structure (42) is stable and does not lead to any fluorescence output in the presence of subunit (41), yet in the absence of strand ND’, curve (a). 12

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The addition of (43) triggers the cleavage of substrate (5), implying that the hairpin was opened, and this led to the self-assembly of the Mg2+-dependent DNAzyme, Figure 9S (D) curve (b). Figure 9S (E) shows the fluorescence intensity generated by the two layer circuit in the absence of the input I8 (a), in the presence of the input I8, but without the amplifying hairpin, (44), (b) , and in the presence of the input I8 and the hairpin, (43), that leads to amplified cleavage of (5), (c). One may realize that the fluorescence intensity in the presence of (44), in a non-optimized configuration of the system, is ca. 35% higher than the intensity generated by the non-amplified system. The concentration of the components shown in Figure 9S (B) are: (5) 1μM; (41) 0.3μM; (42) 0.3μM and N'D1 0.5μM. The concentration of the components shown in Figure 9S (C) are: All of the subunits: 1μM; the hairpin (44): 0.3μM; the caged substrate components (17) and (18) 1μM; the substrate (5) 1μM and the input I8 0.1μM.

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Figure S10 shows the gel electrophoresis results following the YES gate operation.

Figure S10: (A) The caged substrates (15)/(16) and (17)/(18) for the YES gates cascade (the numbers in each colored region reflect the amount of nucleotides in each of the respective regions). (B) Acrylamide electrophoresis of the YES gate system: Control entries: (a) caged substrate (17)/(18) (b) caged substrate (15)/(16) Entries (c) to (f) correspond to the activities of the gate cascade: (c) single YES gate with no input I5 (the caged substrate (15)/(16) is visible). (d) single YES gate activated with the input I5 (one may realize the formation of a new band corresponding to the cleaved substrate).(e) two coupled YES gates with no input. (f) Two coupled YES gates activated with the input I5 (one may realize the formation of the two new bands corresponding to the products of the cleavage of substrates (15)/(16) and (17)/(18), and depletion of the bands corresponding to the substrates (15)/(16) and (17)/(18).

14

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Table S1 depicts the instructions of inputs design and their rules for individual and parallel gates operations.

A

B

C

D

E

Gates

Number of complementary Domain between input-subunits (I1/I2)

Number of complementary Domain Between inputs (I1/I2)

Number of DNAzyme subunits

Number of Substrates

Schematic design of the inputs1 (I1/I2)

OR

2/2

0/0

4

1

AND

1/1

1/1

2

1

XOR

2/2

4/4

4

1

InhibAND

2/0

3/3

2

1

2/2

0/0

2

1

1/1

1/1

2

1

OR+AND

3/3

1/1

6

2

OR+XOR

4/4

4/4

8

2

OR+InhibAND

4/2

3/3

6

2

OR+NOR

4/4

0/0

8

2

OR+NAND

3/3

1/1

6

2

AND+XOR

3/3

4/4

6

2

AND+InhibAND

3/1

3/3

4

2

AND+NOR

3/3

1/1

4

2

AND+NAND

2/2

1/1

4

2

XOR+InhibAND

4/2

5/5

6

2

XOR+NOR

4/4

4/4

6

2

XOR+NAND

3/3

4/4

6

2

NOR+NAND

3/3

1/1

4

2

OR+AND+XOR

5/5

4/4

10

3

InhibAND+AND+XOR (HA+HS)

5/3

5/5

8

3

NAND+AND+XOR

5/5

4/4

8

3

NAND+InhibAND+AND+XOR

5/4

5/5

10

4

NOR

2

NAND

2

1. An and An' represents the domains and their complementary domain, respectively. 2. For the gates the input domains hybridize with a helper nucleic acid that bridges the DNAzyme subunits (see S4 and S5). nature nanotechnology | www.nature.com/naturenanotechnology

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Table S1 summarizes for all individual and set of two, three and four parallel gates: - The number of complementary domains between each of the inputs and the DNAzyme subunits (entry A). - The number of inter-inputs complementary domains between inputs (entry B). - The number of DNAzyme subunits (entry C). -.The number of substrates (entry D). - The schematic design of the inputs (entry E).

The design of the inputs rests on two basic types of complementary nucleic acid sequences: One type includes domains complementary to the respective subunits of the DNAzyme, where the second type includes inter-inputs complementary domain. For the construction of individual and parallel gates, the number of complementary domains is designed using the following instructions:

1. The number of complementary domains (entry A and B) for each individual gates is a mandatory requirement, and these represent the minimums number of domains to activate all of the gates individually.

2. The number of inter-inputs complementary domains between inputs (entry B) is divided into three groups: the AND and NAND, the XOR and InhibAND and the OR and NOR gates. For the first group, the number of complementary domains is only one (as the complementary domains have to stabilize the formation of the duplex between inputs at room temperature, that bridge two DNAzyme subunits into an active DNAzyme). For the second group, the complementary domains need to provide strong hybridization energy, as they destabilize two DNAzyme structures and one DNAzyme structure, for the XOR and for the InhibAND gate, respectively. The third group doesn’t require any of these domains in the inputs sequence. 16

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Following this rules, in order to design parallel gates, the number of inter-inputs complementary domains is a combination of the inter-inputs domains of the individual gates. In fact, upon the activation of a parallel combination of gates from group one and two, the number of inter-inputs complementary domains required to activate these gates, correspond to the number of inter-inputs complementary domains required to activate the individual gate of the second group. For example, in order to activate the AND-XOR gate (HA), the number of

inter-inputs

complementary domains required is 4 in each input, yet this originate from the XOR gate design requirement. Thus, the inter-inputs complementary domains requirement for the AND gate is provide by the design of the XOR gate. On the other hand, upon activating two parallel gates from group two or two parallel gates of group one, one of the inter-inputs complementary domains can be eliminated. For example, by activating the AND-NAND gates, only one inter-inputs complementary domain is required in each of the inputs (the inter-inputs complementary domains requirement to activate both individual gates, can be design in common and have the same function requirement).

3. In order to form and activate a DNAzyme, the bridging of two DNAzyme subunits is mandatory. However, it is possible to use a common DNAzyme subunit to construct and activate two different DNAzyme that will cleave two different substrates. For example, by bridging DNAzyme subunits 1+2 and 1+3, two different DNAzyme will be activated with a common DNAzyme subunit 1. This property is used in the combination and activation of parallel gates. For example by activating the Half- adder and Half-substractor systems (InhibAND-AND-XOR gates), one of the DNAzyme subunits used for the InhibAND and AND gates are identical (the input domain requires for the hybridization with this DNAzyme subunit is also identical for both gates). Nonetheless, the instructions given in this paragraph can be applied only after implementation of the instructions

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given in paragraph (2). This common complementary domain between input-DNAzyme subunit should not be use for the inter-inputs complementary domains design requirement.

S11 Material and method Material- 4-(2-hydroxyethyl)piperazine-1 ethanesulfonic acid sodium salt (HEPES), sodium chloride, Magnesium cloride were purchased from Sigma-Aldrich Inc. DNA oligonucleotides (5, 11, 12, 15, 17, 26 and 29) were purchased from Integrated DNA Technologies Inc. (Coralville, IA). All other oligonucleotide sequences were purchased from Sigma-Genosys. Ultrapure water from NANOpure Diamond (Barnstead) source was used in all of the experiments. DNA sequences: (1) 5' T AGG TAT TTG TAG GTTA CAC CCA TGT TAC TCT 3' (2) 5' GAT ATC AGC GAT TAAC ACT CAG GAT TCG 3' (3) 5' G TGA TGT GTC ATA GTTA CAC CCA TGT TAC TCT 3' (4) 5' GAT ATC AGC GAT TAAC AG TAG TAG TCT GC 3' (5) 5’ FAM-AGA GTA TrAG GAT ATC-Black Hole Quencher-1 3' (6) 5' CGA ATC CTG AGT CTA CAA ATA CCT AG TGA TGT GTC ATA AG TAG TAG TCT GC 3' (7) 5' GC AGA CTA CTA CT TAT GAC ACA TCA CT AGG TAT TTG TAG ACT CAG GAT TCG 3' (8) 5' CTG CTC AGC GAT TAAC AAC TGG TGC TA 3' (9) 5' GA CTC GTA TGC GTTA CAC CCA TGT TAG AGA 3' (10) 5' ATC TGT CGA GTG GTTA CAC CCA TGT TCG TCA 3' (11) 5' Cy5.5-TGA CGA TrAG GAG CAG-Iowa Black RQ 3' (12) 5' ROX-TCT CTA TrAG GAG CAG-Black Hole Quencher-2 3' 18

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(13) 5' CGA ATC CTG AGT CTA CAA ATA CCT AG TGA TGT GTC ATA AG TAG TAG TCT GC ATC TGT CGA GTG GCA TAC GAG TC 3' (14) 5' TAG CAC CAG TT CAC TCG ACA GAT GC AGA CTA CTA CT TAT GAC ACA TCA CT AGG TAT TTG TAG ACT CAG GAT TCG 3' (15) 5' A TAC GCT TAT CGG CAC ATG AGA TCT CTA TrAG GAG CAG GAG TG AA CTG 3' (16) 5' TA GCA TCA GTT CAC TCG ACA GAT TCT CAT GTG CCG ATA AGC GTA T 3' (17) 5' C TGG TCT GGT GCA GCA CTG GTA TGA CGA TrAG GCA AGA TCA TA AG TAG 3' (18) 5' GC AGA CTA CTA CT TAT GAC ACA TCA C TAC CAG TGC TGC ACC AGA CCA G 3' (19) 5' GA CTC GTA TGC GTTA CAC CCA TGT TCG TCA 3' (20) 5' TCT TGC AGC GAT TAAC AAC TGA TGC TA 3' (21) 5' CTG CTC AGC GAT TAAC ACT CAG GAT TCG 3' (22) 5' T AGG TAT TTG TAG GTTA CAC CCA TGT TAG AGA 3' (23) 5' CGA ATC CTG AGT CTA CAA ATA CCT A 3' (24) 5' ATC TGT CGA GTG GCA TAC GAG TC 3' (25) 5' G TGA TGT GTC ATA AG TAG TAG TCT GC 3' (26) 5' A TAC GCT TAT CGG CAC ATG AGA TGA CGA TrAG GCA AGA TGT AG AC TCA 3' (27) 5' CGA ATC CTG AGT CTA CAA ATA CCT A TCT CAT GTG CCG ATA AGC GTA T 3' (28) 5' TA GCA TCA GTT CAC TCG ACA GAT 3'

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supplementary information

doi: 10.1038/nnano.2010.88

(29) 5' GTG GTC CAG TGC TGC AGT GCA ATC TCA TGT ACC GAT AAG CGT AT AGA GTA TrAG GAT ATC GG TTG GTG TGG TTG G 3' (30) 5' CAC CAA CC AT ACG CTT ATC GGT ACA TGA GAT TGC ACT GCA GCA CTG GAC CAC 3' (32) 5' ATC TGT CGA GTG GCA TAC GAG TC 3' (33) 5' TAG CAC CAG TT CAC TCG ACA GAT 3' (34) 5' GTA GTA GTC TGC ATC TGT CGA GTG AA CTG GTG CTA 3' (35) 5' TAG CAC CAG TT CAC TCG ACA GAT GCA GAC TAC TAC 3' (36) 5' ATG ACA CAT CAC ACT CAG GAT T 3' (37) 5' GG TAT TTG TAG GTG ATG TGT CAT 3' (38) 5' ATG CAG ACT ACT ACT CGA ATC CTG AGT CTA CAA ATA CCT ATA TGA CAC ATC ACG AT 3' (39) 5' ACT CAG GAT TCG AGT AGT AGT CTG CAT 3' (40) 5' AT CGT GAT GTG TCA TAT AGG TAT TTG TAG 3' (41) 5' GAT ATC AGC GAT ATT GGT GAG 3' (42) 5' AT TGG TGA TAT GA TC TTG CTC ACC AAT CAC CCA TGT TAC TCT 3' (43) 5' TCT TGC AGC GAT TAA CCA CTC TAT GA TC TTG AGT GGT TA 3'

Instrumentation: Light emission measurements were performed using a photon counting spectrometer (Edinburgh Instruments, FLS 920) equipped with a cooled photomultiplier detection system, connected to a computer (F900 v 6.3 software). The excitation of Cy5.5 (F1), FAM (F2) and ROX (F3) was done at 700 nm, 495 nm and 648 nm, respectively.

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supplementary information

doi: 10.1038/nnano.2010.88

Experimental section: Logic gate systems: All the assays were prepared into 10 mM HEPES buffer containing 1M NaCl and 20 mM

MgCl2. They include 1µM of appropriate DNAzyme subunits,

substrates and 0.9 µM of the appropriate inputs describe in each figures for each state and respective gate. The solution was then heated to 90°C for 5 minutes and cooled instantly to 25°C and holded at this temperature for 1 hour and 30 minutes for simple gate (AND, XOR, InhibAND, NAND and NOR gates) and for the Half adder/ Half substractor systems and 3 hours for the cascade and fan-out systems.

Inhibition of thrombin by a DNA-based machine: The assay was done into a 10 mM HEPES buffer containing 1M NaCl, 20 mM MgCl2 and 7mM KCl. We add 1µM of the appropriate DNAzyme subunits, substrate and 0.9 µM of the input. The solution was then heated to 90°C for 5 minutes and cooled instantly to 25°C and holded at this temperature for for 1 hour and 30 minutes. After that we add 20 nM of Thrombin for half an hour in order to form the aptamers/substrate complex. For the fluorescence measurements chromogenic substrate Rhodamine110 bis(p-tosyl-Gly-Pro-Arg), 5.8x10M-6 was used.

Nondenaturing

polyacrylamide

gel

electrophoresis:

Gels

contained

30%

polyacrylamide (acrylamide/bis-acrylamide). Tris-borate-EDTA (TBE) consisting of Tris base (89 Mm, pH= 7.9), boric acid (89 mM) and EDTA (2 mM) was used as the separation buffer and Gels were run on a Hoefer SE 600 electrophoresis unit at 25°C (300 V, constant voltage) for 3 hours. After electrophoresis, the gels were stained with SYBR Gold nucleic acid gel stain (Invitrogen) and scanned.

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