Molecular Devices

DOI: 10.1002/ange.200502511

Construction of Molecular Logic Gates with aDNA-Cleaving Deoxyribozyme**

Xi Chen, Yifei Wang, Qiang Liu, Zhizhou Zhang,*Chunhai Fan,* and Lin He*

Logic gates are devices that perform the basic logic oper-ations AND, NOT, and OR, as well as their combinations. It iswell-known that electronic logic gates form the basis ofconventional silicon computer microprocessors. By analogy,molecular logic gates are crucial for the development ofmolecular-scale computers, which have attracted significantresearch interest.[1] In particular, nucleic acids (DNA andRNA) have proven to be highly useful building blocks for theconstruction of molecular logic gates and computationaldevices.[2–8] Herein, we report the construction of molecularlogic gates that are made entirely of DNA molecules. Asexamples, we demonstrate the performance of logic oper-ations including “YES”, “NOT”, and a three-input gate

“AND(A,NOT(B),NOT(C))” through the recognition andcatalytic properties of DNA. Notably, the combination of the“NOT” gate and the “AND(A,NOT(B),NOT(C))” gate can,in principle, make a universal operator set.

Artificially designed DNA or RNA sequences can foldinto well-ordered, three-dimensional structures that eitherrecognize corresponding ligands (aptamers)[9] or catalyzespecific chemical reactions (deoxyribozymes or ribo-zymes).[10–21] In their pioneering work, Stojanovic and co-workers constructed molecular logic gates based on in vitroselected RNA-cleaving deoxyribozymes (DNAzymes) byexploiting allosteric regulation.[6–8] In their system, theactivities of deoxyribozymes were allosterically regulated byspecific effectors, which were rationally designed oligonu-cleotides containing complementary sequences to targetdeoxyribozymes. Either the presence or the absence ofeffectors was employed as the input, while intact or cleavedsubstrates were regarded as outputs. The same group laterconstructed complex systems based on this elegant strategy,such as a molecular-scale half-adder[7] and a MAYA autom-aton that could play a tic-tac-toe game with a humanadversary.[8]

Despite the elegance of this strategy, these logic gatesremain to be improved. In particular, as they are based onRNA-cleaving deoxyribozymes, the corresponding substratesmust be either RNA or chimeric DNA (a piece of oligode-oxynucleotide containing at least one ribonucleotidebase).[6–8] This prerequisite leads to high synthesis costs andsusceptibility to oligonucleotide degradation. In view of thisfact, it is highly desirable to develop ribonucleotide-freemolecular logic gates. Herein, we employ a Cu2+-dependentDNA-cleaving deoxyribozyme as the building block for theconstruction of robust molecular logic gates that are based onDNA only. Our inputs (effectors), gates (deoxyribozymes),and outputs (substrates) are all inexpensive, chemically stableDNA oligonucleotides (Figure 1). The Cu2+-dependentDNA-cleaving deoxyribozyme, which was first isolated byBreaker and co-workers,[22] can catalytically cleave DNAsubstrates by oxidative modification of DNA, which involvesthe generation of hydroxyl radicals through metal-ion-medi-ated redox reactions.[22–24]

Figure 1. The Cu2+-dependent deoxyribozyme and logic gates. A) Struc-ture and consensus sequence of Cu2+-dependent deoxyribozyme forDNA substrate cleavage. R, Y, and N represent purine, pyrimidine, andany nucleotide, respectively. The triangle identifies the cleavage site ofthe substrate. B) Deoxyribozyme as the building block of logic gatesand its corresponding substrate. FAM= fluorescein dye.

[*] X. Chen, Y. Wang, Q. Liu, Prof. Dr. Z. Zhang, Prof. Dr. C. Fan,Prof. Dr. L. HeBio-X Life Science Research CenterShanghai Jiao Tong University, Shanghai 200030 (China)Fax: (+86)21-6282-2491E-mail: zhangzz@sjtu.edu.cn

fchh@sinap.ac.cnhelin@sjtu.edu.cn

Prof. Dr. C. FanShanghai Institute of Applied PhysicsChinese Academy of Sciences, Shanghai 201800 (China)Fax: (+86)21-5955-6902

Prof. Dr. Z. ZhangTianjin University of Science and TechnologyTianjin 300040 (China)Fax: (+86)22-6027-2071

[**] This work was supported by Shanghai Municipal Commission forScience and Technology (Grant Nos. 03DZ14025, 02JC14029,0452m068, 60537030), the Shanghai Rising-Star Program, NationalNatural Science Foundation (20404016), the National “863” Project(Grant No. 2003AA226011), the Key Projects of the Ministry ofEducation (No. 03066), and the Chinese Academy of Sciences.

Supporting information for this article is available on the WWWunder http://www.angewandte.org or from the author.

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We first constructed a “YES” gate (positive sensor) byemploying a deoxyribozyme, DNAzyme_X, with a positive-regulation module (Figure 2, top left). In this design, twomodules (oligonucleotides) were appended to either the 3’ or

5’ end of the hammerhead-like deoxyribozyme (see Support-ing Information). The distal module was complementary tothe catalytic core while the proximal module served as theflexible loop (loop I). In the absence of the effector EX,which contained a complementary sequence to loop I (T7–G26), the distal module formed a stem (stem I) with thecatalytic core. This stem formation disrupted the structure ofthe catalytic core and set DNAzyme_X at the “OFF” state. Inthe presence of the effector EX, the binding of EX to loop Iprevented the formation of stem I, thus turning the DNAzy-me_X to the “ON” state. As demonstrated in Figure 2A andin the Supporting Information, the activity of DNAzyme_Xwas effectively regulated by the effector EX, with theapparent catalytic reaction rate constant kobs at ca. 0.1 min�1

of the “ON” state and ca. 0.01 min�1 of the “OFF” state (ON/OFF ratio 10:1). This ON/OFF ratio, while already sufficientfor demonstration of the “YES” gate effect, might be furtherimproved by appending distal modules to both ends, whichshould significantly reduce the kobs value of the “OFF”state.[25] Notably, the effector binds to the catalytic core in this“YES” gate design, instead of to the substrate-recognitionregion, which makes it feasible to realize multiple turnovers.

This important feature should be applicable to the design ofother molecular logic gates, and might lead to significantimprovement in the kobs value of the “ON” state.

We then constructed a “NOT” gate (negative sensor) byusing DNAzyme_Y (Figure 2, center left) which contained anegative-regulation module (loop II, A20–C34). Note that thetriplex-forming stem-loop was essential for DNAzyme_Y (seeSupporting Information).[23,24] In the absence of the effec-tor EY, the deoxyribozyme was active (“ON” state) as thetriplex was intact. In contrast, in the presence of the effector,EY bound to loop II, which competitively opened stem II anddisrupted triplex formation. This process effectively inhibitedthe enzyme activity and set the DNAzyme_Y at the “OFF”state. With this “NOT” gate, we observed that the kobs of the“ON” state was ca. 0.05 min�1, whereas that of the “OFF”state was ca. 0.001 min�1, which indicates that the ON/OFFratio of this “NOT” gate reached 50:1 (Figure 2B andSupporting Information).

The “NOT” gate can also be constructed by introducing a“pistol-like” structure of the deoxyribozyme (Figure 2,bottom left). It was reported that the two deoxyribonucleo-tide moieties of the 3’ terminus, “AC”, were conserved duringin vitro selection,[23] which suggests that these two deoxyri-bonucleotides are crucial for catalysis. In spite of this, partiallytruncated deoxyribozymes (in the absence of 3’-terminal C)were reported to be active in the presence of both Cu2+ ionsand ascorbate.[24] We then designed a deoxyribozyme, DNA-zyme_Z, which had an extended sequence at the 3’ terminus(see Supporting Information). We employed five potentialeffectors, each being partially complementary to DNA-zyme_Z (EZ: T32–T46, EZ-G: C31–T46, EZ-GT: A30–T46,EZ-GTT: A29–T46, and EZ-GAA: C31–T46). By individu-ally analyzing the deoxyribozyme activity in the presence ofboth Cu2+ and ascorbate, we observed that pairing of theeffector with C31 had little effect on the catalytic activity,pairing with both C31 and A30 reduced the catalytic activityto some extent, and pairing with C31, A30, and A29 stronglyinhibited the deoxyribozyme (data not shown). Therefore, weselected the strongest inhibitor EZ-GTT as the effector forthe “NOT” gate. Interestingly, when DNAzyme_Z wastreated with EZ-GTT, the initial kobs value was close tozero. However, we observed that the rate increased with theincubation time (Figure 2C and Supporting Information).Given that the effector was located at the catalytic core ofDNAzyme_Z, the increase of kobs might be caused by thedestruction of the effector and/or the negative-regulationmodule. In spite of this apparent time-dependent change ofactivity at the “OFF” state, the signal-to-noise ratio in the first20 minutes of the reaction was sufficient for its function as a“NOT” gate (ON/OFF ratio 10:1), that is, DNAzyme_Z wasat the “ON” state in the absence of the effector EZ-GTTandat the “OFF” state in the presence of the effector.

On the basis of this “NOT” gate with the pistol-likestructure, we further designed an A6:B6:C gate (AANDNOT B ANDNOT C, or in the prefix form, AND-(A,NOT(B),NOT(C))). As far as we know, such a DNA-based logic gate has not been previously reported. Bydefinition, the logic gate A6:B6:C is at the “ON” statewhen and only when input A is “1”, input B is “0”, and

Figure 2. Left: Secondary structures of DNAzymes: DNAzyme_X(“YES” gate), DNAzyme_Y (“NOT” gate I), and DNAzyme_Z (“NOT”gate II). Schematic drawings and detailed descriptions of the transitionbetween the “ON” and “OFF” states for these three gates are shownin the Supporting Information. Right: Plots of the reaction time versusfraction of cleaved products (“ON” (*), “OFF” (^); the correspondingPAGE pictures are shown in the Supporting Information). A) DNA-zyme_X (“YES” gate); B) DNAzyme_Y (“NOT” gate I); C) DNAzyme_Z(“NOT” gate II).

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input C is “0”. We then proposed that DNAzyme_XYZ(Figure 3A) could function as the logic gate A6:B6:C bydefining the presence of effectors as input 1 and their absenceas input 0. We examined the catalytic activity of DNA-zyme_XYZ in either the absence or presence of one or

several of the three effectors (23= 8 combinations). Asexpected, after 20 min reaction time the cleaved productwas only observed when the deoxyribozyme was treated withEX, while the absence of both EY and EZ-GTT inducedsubstrate cleavage (Figure 3B). We thus successfully demon-strated the construction of a DNA-based three-input logicgate based on simple, basic logic gates. Importantly, this novelgate may form the basis of completely DNA-based half-adders[7] or automata.[8]

The proposed logic gate system based on DNA-cleavingdeoxyribozyme has several inherent advantages. First, incontrast to RNA or chimeric DNA, the synthesis of DNAoligonucleotides is fairly inexpensive. Second, unlike chimericDNA that can only be chemically synthesized, DNA strandsmight be produced by the PCR, which is much moreconvenient in molecular biology laboratories. Third, oursystem is completely DNA-based, thus obviating the specialprecautions required in the use of RNase which may induceunexpected substrate degradation. Fourth, the speed of oursystem matches or even exceeds that of the previouslyreported sequence-specific nucleic acid sensing systemsbased on RNA-cleaving deoxyribozymes (kobs> 0.01 min�1

at the “ON” state). In addition, this Cu2+-dependent deox-yribozyme might inspire many other interesting applications.For example, products of oxidative cleavage carry 3’-terminal-modified phosphates, which are resistant to 3’ to 5’ exonu-cleases.[24] This means that these products are potentiallyinsusceptible to the restriction–modification system in livingorganisms. This feature may allow the design of a program-

med DNA modification system in which the modificationability comes from the oxidative cleavage activity of deoxy-ribozymes.

In view of these advantages of our logic-gate system, thesequences of the deoxyribozymes involved remain to befurther optimized. Notably, the gate activities of oligonuclo-tides with minor sequence differences can vary by as much asseveral orders of magnitude. Other possible disadvantages areas follows: First, the optimal conditions for the oxidativecleavage reaction of deoxyribozymes are different fromphysiological conditions,[24] and thus the logic gates describedherein are not expected to work in vivo in their present form.Second, deoxyribozymes are known to undergo self-cleavage.The self-cleavage rate is significantly lower than that ofsubstrate cleavage,[24] but this still makes it difficult to achievemultiturnover, which is a useful feature for the design ofbiosensors with amplified signals. These problems still remainto be explored.

In summary, we have described the construction of DNA-based molecular logic gates through modular design. Incontrast to all previously reported ribonucleotide-containingsystems, the employment of a Cu2+-dependent DNA-cleavingdeoxyribozyme enabled us to design the highly robust logicgates “YES”, “NOT”, and A6:B6:C solely with DNAmolecules. This strategy should be potentially useful for theconstruction of any DNA-based molecular logic gates andcomputing devices that are essentially free from RNasedigestion.

Experimental SectionOligonucleotides and reagents: The oligonucleotides were obtainedfrom Bioasia (Shanghai) and purified by high-pressure liquidchromatography (HPLC). Stock solutions of oligonucleotides (10–40 mm) were stored at �20 8C. The sequences of the effectors EX andEY were 5’-CTACTATTCAATTCCGTTAA and 5’-CACTGGACA-TAACTT, respectively. The sequences of the other oligonucleotidesare shown in the figures. The secondary structures of the oligonu-cleotides were examined by using Zuker and TurnerGs “mfold” DNA-folding site (http://www.bioinfo.rpi.edu/applications/mfold/old/dna/).The concentrations of the oligonucleotides were measured by UVabsorption at 260 nm. l-Ascorbate (Ameresco) was stored in aliquots(100 mm) at �20 8C.

Reaction conditions: Reactions were carried out at 23 8C in thepresence of 5’-FAM tagged substrate (250 nm), deoxyribozyme(250 nm), effector (10 mm), HEPES buffer (50 mm, pH 7.0), NaCl(0.3m), CuCl2 (10 mm), and l-ascorbate (10 mm). Note that althoughthis deoxyribozyme exhibits significant catalytic activity in thepresence of Cu2+ as the only cofactor,[23] the co-existence of Cu2+

and ascorbate further facilitates the DNA cleavage reaction.[24]

Before starting the catalytic reaction, deoxyribozymes and effectors(if used) were mixed in the reaction buffer, heated to 90 8C for 1 min,and then slowly cooled to room temperature during a 30-min period.The substrates were then added to the solution and the reaction wasinitiated by the addition of both CuCl2 and l-ascorbate.

Kinetic analysis: Aliquots were removed at appropriate intervalsand quenched with the stop buffer (40 mm EDTA, 8m urea, 89 mm

Tris base, 89 mm boric acid, 20% sucrose, and 0.5% bromophenolblue). Gel electrophoresis was carried out with a Bio-Rad Mini-Protean II cell, and a fluorescence image was obtained and quantifiedby a Kodak Image Station 2000 MM with ImageQuant (Molecular

Figure 3. Three-input logic gate A6:B6:C. A) Secondary structure ofDNAzyme_XYZ. B) Products and precursors were analyzed by 12%denatured PAGE after 20 min reaction. The employed effectors are thesame as those described in Figure 2. The symbols + and � stand forthe presence (input 1) and absence (input 0) of effectors, respectively.“Uncleaved” and “cleaved” indicate intact and cleaved products,respectively. Note that the cleaved product (output) is only observedin the presence of EX and in the absence of both EY and EZ-GTT.

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Dynamics). The kobs value was determined from the slope of initialvalues in plots of cleaved fraction versus time.

Received: July 19, 2005Revised: November 21, 2005Published online: February 10, 2006

.Keywords: biosensors · DNA · molecular devices · ribozymes ·RNA

[1] A. Saghatelian, N. H. Volcker, K. M. Guckian, V. S.-Y. Lin,M. R. Ghadiri, J. Am. Chem. Soc. 2003, 125, 346.

[2] L. M. Adleman, Science 1994, 266, 1021.[3] Q. Liu, L. Wang, A. G. Frutos, A. E. Condon, R. M. Corn, L. M.

Smith, Nature 2000, 403, 175.[4] Y. Benenson, T. Paz-Elizur, R. Adar, E. Keinan, Z. Livneh, E.

Shapiro, Nature 2001, 414, 430.[5] A. Okamoto, K. Tanaka, I. Saito, J. Am. Chem. Soc. 2004, 126,

9458.[6] M. N. Stojanovic, T. E. Mitchell, D. Stefanovic, J. Am. Chem.

Soc. 2002, 124, 123.[7] M. N. Stojanovic, D. Stefanovic, J. Am. Chem. Soc. 2003, 125,

6673.[8] M. N. Stojanovic, D. Stefanovic, Nat. Biotechnol. 2003, 21, 1069.[9] S. E. Osborne, A. D. Ellington, Chem. Rev. 1997, 97, 349.[10] K. Kruger, P. J. Grabowski, A. J. Zaug, J. Sands, D. E. Gottsch-

ling, T. R. Cech, Cell 1982, 31, 147.[11] C. Guerrier-Takada, K. Gardiner, T. Marsh, N. Pace, S. Altman,

Cell 1983, 35, 849.[12] J. Tang, R. R. Breaker, Chem. Biol. 1997, 4, 453.[13] G. A. Soukup, R. R. Breaker, Curr. Opin. Struct. Biol. 2000, 10,

318.[14] M. P. Robertson, A. D. Ellington, Nat. Biotechnol. 1999, 17, 62.[15] M. P. Robertson, A. D. Ellington, Nucleic Acids Res. 2000, 28,

1751.[16] M. Araki, Y. Okuno, Y. Hara, Y. Sugiura, Nucleic Acids Res.

1998, 26, 3379.[17] D. Y. Wang, B. H. Lai, A. R. Feldman, D. Sen,Nucleic Acids Res.

2002, 30, 1735.[18] D. H. Burke, N. D. Ozerova, M. Nilsen-Hamilton, Biochemistry

2002, 41, 6588.[19] Y. Komatsu, S. Yamashita, N. Kazama, K. Nobuoka, E. Ohtsuka,

J. Mol. Biol. 2000, 299, 1231.[20] T. Kuwabara, M. Warashina, K. Taira, Trends Biotechnol. 2000,

18, 462.[21] M. N. Stojanovic, P. de Prada, D. W. Landry, ChemBioChem

2001, 2, 411.[22] N. Carmi, L. A. Shultz, R. R. Breaker, Chem. Biol. 1996, 3, 1039.[23] N. Carmi, S. R. Balkhi, R. R. Breaker, Proc. Natl. Acad. Sci.

USA 1998, 95, 2233.[24] N. Carmi, R. R. Breaker, Bioorg. Med. Chem. 2001, 9, 2589.[25] V. Saksmerprome, D. H. Burke, Biochemistry 2003, 42, 13879.

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