dna computing circuits using libraries of dnazyme subunits

DNA computing circuits using librariesof DNAzyme subunitsJohann Elbaz1, Oleg Lioubashevski1, Fuan Wang1, Francoise Remacle2, Raphael D. Levine1

and Itamar Willner1*

Biological systems that are capable of performing compu-tational operations1–3 could be of use in bioengineering andnanomedicine4,5, and DNA and other biomolecules havealready been used as active components in biocomputationalcircuits6–13. There have also been demonstrations ofDNA/RNA-enzyme-based automatons12, logic control of geneexpression14, and RNA systems for processing of intracellularinformation15,16. However, for biocomputational circuits to beuseful for applications it will be necessary to develop alibrary of computing elements, to demonstrate the modularcoupling of these elements, and to demonstrate that thisapproach is scalable. Here, we report the construction of aDNA-based computational platform that uses a library of cata-lytic nucleic acids (DNAzymes)10, and their substrates, for theinput-guided dynamic assembly of a universal set of logic gatesand a half-adder/half-subtractor system. We demonstrate mul-tilayered gate cascades, fan-out gates and parallel logic gateoperations. In response to input markers, the system can regu-late the controlled expression of anti-sense molecules, or apta-mers, that act as inhibitors for enzymes.

Our biocomputing approach is based on two libraries of nucleicacids, one consisting of subunits of DNAzymes and the second theirsubstrates (Fig. 1a, boxes I and II). In the presence of the appropriatenucleic acid inputs, the simultaneous selection of pre-designedDNAzyme subunits and substrates from these respective librariesresults in the assembly of the computational unit (box III) in twomodules. The ‘input module’, which controls the gate functionality,consists of the input strands and ‘recognition arms’ of theDNAzyme subunits, and the ‘processing module’ includes the cata-lytic DNAzyme core that binds to the substrates. The input-guidedassembly of the gate unit results in the cleavage of the substrate andreleases the product strand. Thus, the independent structures of theinput and processing modules provide diversity and modularity inthe 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 gatefunctionality. Also, in most of those systems1,7–9, the structuresoperated as single-use gates. In contrast, we introduce a new gateadaptation principle in which the inputs guide the assembly andfunctionality of the gates through the selection of the correspondingsubunits. The versatility of our method lies in the ability to selectindividual subunits to assemble different gates, and also in thefact that the gates are non-destructible.

DNAzymes17,18 are of growing interest in the fields of DNAnanotechnology19, bio-analysis20,21 and targeted therapy22,23.TheMg2þ-dependent E6-type DNAzyme24 catalyses hydrolytic cleavageof a ribonucleobase (rA)-containing DNA substrate with a catalyticrate of 0.01 min21 (Fig. 1b). The use of DNAzyme subunits to

construct the computing elements is shown and explained inFig. 1c,d. The construction of the XOR gate is outlined in Fig. 2a.The system consists of the DNAzyme subunits (1)–(4), the fluoro-phore/quencher-labelled substrate (5), and the inputs I1 and I2(6) and (7). In the presence of I1 or I2, two different DNAzymestructures are formed, leading to the cleavage of the mutual sub-strate and to the generation of fluorescence (‘True’ output).Triggering the system with both inputs, I1 and I2, results in the for-mation of the energetically favoured duplex between (6) and (7)(DG8≈ 260 kcal mol21, compared to DG8≈ 214 kcal mol21 forthe duplex structures between the inputs and the DNAzyme sub-units, where DG8 correspond to the free energy changes upon theformation of the respective duplexes). This prohibits the formationof the active DNAzyme and the generation of fluorescence signals(‘False’ output). The experimental results of this system are pre-sented in Fig. 2b, showing that fluorescence is triggered only uponactivation by I1 or I2 alone, and is extinguished in the presence ofI1 and I2, consistent with the operation of a XOR gate (for kineticresults, 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) andhalf-subtractor (HS) systems in a single test tube (Fig. 2c). An HAdevice produces Sum and Carry outputs, whereas an HS producesDifference and Borrow outputs. Parallel activation of the HA andHS devices requires the implementation of the AND, XOR andInhibAND gates in a single test tube. This was demonstrated byapplying the same two inputs (I3 and I4) in a system with alibrary of DNAzyme subunits (1)–(4) and (8)–(10) and a collectionof substrates (12), (5) and (11), each labelled with a different fluor-ophore (F1, F2, F3). Inputs I3 and I4 select the respective DNAzymesubunits from the library. Each of the fluorophores generates theoutput of a single gate (F1¼AND; F2¼ XOR; F3¼ InhibAND).The fluorescence intensities corresponding to the output of thethree gates are presented in Fig. 2d. The Carry bit output (ANDgate) is probed by F1, l¼ 610 nm, and is observed only upon inter-action with both inputs (I3, I4). The Sum and Difference bits (givenby the same XOR gate) are presented by F2, l¼ 520 nm, whichgives a True output in the presence of either I3 or I4, but a Falseoutput upon triggering by both inputs, due to preferred inter-input hybridization. The Borrow bit output (InhibAND gate),given by F3, l¼ 710 nm, corresponds to a True value in the pres-ence of I4 only. The fluorescence values resulting from the paralleloperation of the HA and HS computation modules and the corre-sponding truth tables are presented in Fig. 2e. (For electrophoresisexperiments supporting the AND gate, see Supplementary Fig. S6.)

Challenges in DNA computing include achieving communi-cation between different devices and scalability of the device

1The Institute of Chemistry, The Hebrew University of Jerusalem, Jerusalem 91904, Israel, 2Chemistry Department, B6c, University of Liege, 4000 Liege,Belgium. *e-mail: [email protected]

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modules, and elimination of leakage and crosstalk. To this end, wehave designed a caged substrate in which the output strand is pro-tected until its release by the input-assembled DNAzyme.DNAzyme-stimulated cleavage of the substrates yields nucleic acidoutputs that act as inputs for activating gates cascades or fan-outgates, thus providing multilayer circuits. Figure 3a depicts the con-figuration of the caged substrate, which consists of a ribonucleobase-containing sequence (17) that is partially hybridized with thenucleic acid (18) in regions II and III. Region I contains theDNAzyme cleavage site TrAGG, and the complementary domainsacting as the substrate for the DNAzyme. Region II includes basesequences C and D, which provide the input for the subsequentgate. The protected sequence C–D is inactive before cleavage ofthe substrate, because the duplex (17)/(18) is energetically favoured(DG ≈ 240 kcal mol21) compared to the duplex of C–D with theDNAzyme subunits of the next layer (DG ≈ 214 kcal mol21).Note that duplex III in (17)/(18) is longer than duplex II (22 basepairs, compared to 10 base pairs) and duplex III, therefore, providesstabilization for blocking domain C–D in the caged substrate. In thepresence of the appropriate input and DNAzyme subunits, thecaged substrate is cleaved in region I, synergetic stabilization of

the two duplex domains (regions II and III) is eliminated, and theprotected sequence C–D is released. The single-stranded nucleicacid (C–D) then acts as an input for the subsequent gate.Although for the gate cascade the cleavage domains and the pro-tected output sequences are different (Fig. 3b), the caged substratesfor the fan-out gates include identical cleavage domains, but differ-ent protected output sequences (Fig. 3c). Figure 3d presents acascade of YES2AND2InhibAND gates using a library ofDNAzyme subunits (3), (4) and (19)–(22), the caged substrates asduplexes (15)/(16) and (17)/(18), the fluorophore-quencher-func-tionalized substrate (5), and inputs I5 (23), I6 (24) and I7 (25). Inthe presence of input I5 (23), the appropriate DNAzyme subunits(21) and (22) are selected from the library and assemble the YESgate with caged substrate (15)/(16). The cleaved substrate yieldsthe strand R1EF, which acts as input for the AND gate. Althoughstrand R1EF alone cannot form any active DNAzyme with thelibrary components, in the presence of the second input I6 (24),the DNAzyme structure corresponding to the AND gate isformed. This results in the cleavage of substrate (17)/(18) and therelease of strand R2CD, which, together with the auxiliary inputI7 (25), act as inputs for the third-layer InhibAND gate. Although

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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.

LETTERS NATURE NANOTECHNOLOGY DOI: 10.1038/NNANO.2010.88

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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 F2¼ Fluorescein, lmax¼ 520 nm; AND gate,

emission of F1¼ ROX, lmax¼ 610 nm; InhibAND gate, emission of F3¼Cy5.5, lmax¼ 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 DOI: 10.1038/NNANO.2010.88 LETTERS





I7 (25) cannot generate any active DNAzyme, the released strandR2CD assembles the active DNAzyme, leading to the cleavage of(5) and resulting in fluorescence. In the presence of the twoinputs, formation of duplex R2CD/I7 is favoured, and the formation

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

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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.






of the library components, no cleavage of (5) (and formation of thefluorescent signal) occurred. Using a similar concept, theYES2YES2YES gate cascade was assembled (for results, seeSupplementary Figs S7–S9). The DNAzyme-induced ‘metabolic’cleavage of the substrate was then applied to fan-out the YESgates (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 byinput I8 (EF), leading to the cleavage of the two substrates(17)/(18) and (26)/(27) and yielding the outputs R3AB andR2CD, which act as inputs for two parallel YES gates that activatethe cleavage of substrates (12) and (5), respectively (Fig. 4b). Theactivation of serial or parallel gates through the metabolic cleavageof the caged substrates represents a major advance in biocomputing,because the DNAzyme-based cascaded gates demonstrate scalability

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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 lmax¼ 510 nm; YES gate 3, emission lmax¼ 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).

NATURE NANOTECHNOLOGY DOI: 10.1038/NNANO.2010.88 LETTERS





of the circuits. One should realize, however, that the inputsgenerated by the gate cascades, as expected, weaken with increasingdepth of the circuit. Furthermore, the non-stoichiometric gener-ation of inputs and required inter-input hybridization (1,1 state)might hamper operation of the cascade. Nonetheless, the presentedapproach allows tailoring of an appropriate substrate for amplifica-tion of the gate output by a feedback mechanism (SupplementaryFig. S10). This path enables tuning of the content of the releasedoutput, relatively to the other gates, as well as amplification of theoutput. It should be noted that, by appropriately designingthe inputs, all individual gates and all parallel gates can beconstructed. In fact, for parallel gates, the inputs consist of a com-bination of sequence domains that correspond to the inputs of theindividual gates. (For instructions regarding input design see theSupplementary Information, Discussion and Table S1.)

The resulting constructs of logic gates translate input strands intopre-designed nucleic acid outputs. Thus, DNAzyme-based logicdevices may contribute to the design of biotherapy methods,where input biomarkers are translated into anti-sense or proteininhibitors, released as outputs. This may be exemplified by a YESgate that yields the thrombin-binding aptamer (TBA) using anucleic acid input, as a model for the biomarker. Thrombin is ahydrolytic enzyme participating in blood clotting, and high levelsof thrombin are generated following brain haemorrhage ortrauma, causing damage to brain cells or oedema25. The TBA con-sists of a G-rich 15-mer nucleic acid that self-assembles into aG-quadruplex that binds to thrombin and inhibits its activity26.The input-stimulated release of TBA is described in Fig. 4d. TheDNAzyme subunits (1) and (2) and the caged substrate consistingof duplex (29)/(30), which includes the caged TBA sequence,form the components of the system. Upon activation of thesystem with input I5, the substrate is cleaved, and the releasedTBA inhibits the hydrolytic activity of the thrombin. This activitywas probed by the cleavage of the Rhodamine 110-labelled arginine-containing peptide (31), which triggers the fluorescence of peptidede-protected fluorophore. Figure 4e shows that although the non-inhibited thrombin effectively yields the fluorophore, the input-stimulated release of the aptamer inhibits the proteolytic activity ofthe thrombin, and only �40% of the enzyme activity is retained.Thus, the logic system demonstrates control of enzyme activity. Infact, any biomarker may activate a YES gate and be translated intoan input sequence that activates the DNAzyme computation system.

In conclusion, the present study has introduced a new protein-free biocomputing platform based on a library of DNAzyme sub-units, pre-designed substrates and instructive inputs. The unique-ness of the method lies in the modularity of the gate construct,the input-guided assembly of computing circuits, and the fact thatcomputing elements are non-degradable following operation ofthe gates. We demonstrate the construction of a universal set oftwo-input logic gates leading to HA and HS computationalmodules in a single test tube. Furthermore, the study has demon-strated the modularity and scalability of the computing elements,by constructing multilayered logic circuits and executing gatemultiplication with a fan-out scheme. As we have constructed auniversal set of logic gates, any Boolean logic unit may be assembledby the appropriate combination of gates. Finally, we discussedthe use of the DNAzyme-based computing elements for futurenanomedicine applications.

Received 9 October 2009; accepted 7 April 2010;published online 30 May 2010; corrected after print9 February 2011

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AcknowledgementsParts of this research are supported by the EC Project MOLOC and by the Office ofNaval Research, USA. F.R. is Director of Research at Fonds National de la RechercheScientifique (FNRS), Belgium. J.E. acknowledges a Converging Technologies Fellowship(Israel Science Foundation).

Author contributionsJ.E. designed the systems, performed the experiments, analysed the results and participatedin the formulation of the paper. O.L. participated in designing the system, discussing theresearch results and the formulation of the paper. F.W. participated in designing the systemand performed the experiments. R.D.L and F.R. participated in discussing the researchresults and the formulation of the paper. I.W. supervised the project, evaluated the researchresults and participated in the formulation of the paper.

Additional informationThe authors declare no competing financial interests. Supplementary informationaccompanies this paper at www.nature.com/naturenanotechnology. Reprints andpermission information is available online at http://npg.nature.com/reprintsandpermissions/.Correspondence and requests for materials should be addressed to I.W.





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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.

DNA computing circuits using libraries of DNAzyme subunitsJohann 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.

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dna computing circuits using libraries of dnazyme subunits

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