Functional DNA nanotechnology: emerging applications of DNAzymes and aptamers

Yi Lu and Juewen Liu

In the past 25 years, DNA molecules have been utilized both as powerful synthetic building blocks to create nanoscale architectures and as versatile programmable templates for assembly of nanomaterials. In parallel, the functions of DNA molecules have been expanded from pure genetic information storage to catalytic functions like those of protein enzymes (DNAzymes) and specic binding functions like antibodies (aptamers). In the past few years, a new interdisciplinary eld has emerged that aims to combine functional DNA biology with nanotechnology to generate more dynamic and controllable DNA-based nanostructures or DNA-templated nanomaterials that are responsive to chemical stimuli.
Addresses Department of Chemistry, Beckman Institute for Advanced Science and Technology, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA Corresponding author: Lu, Yi (yi-lu@uiuc.edu)

can be deposited to form well-dened patterns [8,9]. Alternatively, DNA has been conjugated to inorganic nanoparticles, which can then be assembled in a programmable manner to form structures containing either a limited number of particles or cross-linked nanoparticle aggregates [1,2,10,11]. These research efforts have demonstrated the power of DNA as a structural molecule, a scaffold, and as a template in developing nanotechnology. Almost in parallel with the development of DNA nanotechnology, the chemical functions of DNA have been expanded beyond the DNA double helix. Since the early 1990s, many DNA molecules known as DNA aptamers have been isolated that are able to bind a broad range of molecules with high afnity and specicity [12,13]. The molecules that can be recognized by aptamers range from small organic molecules to proteins, cells and even intact viral particles [14]. A further advance in the development of function DNAs was made in 1994, when DNA was shown to act as a catalyst for the rst time [15]. Herein, these catalytic DNA molecules are called DNAzymes (also described as DNA enzymes, deoxyribozymes or catalytic DNA elsewhere). Moreover, DNAzymes and aptamers have been combined to form allosteric DNAzymes or aptazymes. These DNAzymes, aptamers and aptazymes are collectively called functional DNAs, whose functions extend beyond the WatsonCrick base pair recognition of complementary strands [16]. Given the tremendous progress made in both DNA nanotechnology and in the study of functional DNA, it is natural to combine these two exciting elds to create a new interdisciplinary eld that uses functional DNA to control and ne-tune the structure and dynamics of DNA nanostructures and materials. This is exactly what has happened in the past few years [17]. Because new DNA functions always involve interactions with other chemical and biological molecules, they allow the generation of smart nanoscale architectures; the assembly and function of these architectures are responsive to chemical or biological stimuli, in much the same way that biomaterials are made and function in cells. Compared with DNA nanotechnology based on base-pair hybridizations, nanotechnologies based on functional DNA could potentially be more versatile and dynamic. The change in chemical or physical properties of the devices can be used to detect those stimuli in a highly sensitive and selective manner. The sensing applications of functional DNA have been recently reviewed [17,18] and this review mainly focuses on recent progress in other aspects of functional DNA nanotechnology.
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Current Opinion in Biotechnology 2006, 17:580588 This review comes from a themed issue on Chemical biotechnology Edited by Jonathan S Dordick and Amihay Freeman Available online 23th October 2006 0958-1669/$ see front matter # 2006 Elsevier Ltd. All rights reserved. DOI 10.1016/j.copbio.2006.10.004

Introduction
Traditionally, DNA molecules were thought to have the sole function of carrying and passing genetic information from one generation to another. About 25 years ago, however, DNA began to nd a new role in the eld of materials science [16]. Structurally, DNA can bind specically to another DNA strand of complementary sequence, with the double-stranded DNA forming a solid rod of up to 50 nm in length. Moreover, DNA can be modied with a wide range of uorophores and other functional groups that enable it to conjugate with nanoparticles. Compared with RNA and proteins, DNA molecules are less susceptible to hydrolysis and thus are highly stable. These attributes make DNA a special biopolymer with highly predictable sequence-dependent properties, and these properties have been exploited to construct DNA-based geometric and topologic structures [4,7]. By using sticky ends, dispersed three- or four-way DNA junctions and other DNA building blocks have been connected into large periodic structures, upon which nanoparticles or proteins
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DNAzymes in nanotechnology
General considerations

DNAzymes are DNA-based biocatalysts capable of performing chemical transformations [15,1922]. These catalysts have not been found in nature, and all known DNAzymes were isolated by in vitro selection. So far, most of their substrates have been found to be nucleic acids. DNAzymes can therefore provide additional control over nucleic-acid-based nanodevices and, because DNAzymes often catalyze multiple turnover reactions, such devices can have amplication effects. Among the many classes of DNAzymes, RNA-cleaving DNAzymes
Figure 1

are the most widely used, mainly because of their simple reaction conditions, fast turnover rates, and signicant modications on their substrate lengths.
DNAzyme-templated material assembly

Liu and Lu [17,23,24,25,26,27,28] were the rst to employ DNAzymes to obtain functional nanomaterials that are sensitive to chemical stimuli. Their work employed the use of a Pb2+-specic DNAzyme (shown bound to its substrate DNA in Figure 1a). In the presence of Pb2+, the DNAzyme (enzyme strand; green) cleaves the substrate strand (black) into two pieces (Figure 1b). In

DNAzyme-templated material assembly. (a) Secondary structure of the Pb2+-specific DNAzyme discussed in the text. (b) Cleavage of the substrate by the DNAzyme in the presence of Pb2+. Pb2+-directed assembly of gold nanoparticles by the DNAzyme when nanoparticles are aligned (c) in a head-to-tail [23] or (d) a tail-to-tail manner [25]. The nanoparticles are depicted in red or blue; a red to blue/purple color transition is observed on assembly, owing to surface plasmon effects. (e) For head-to-tail aligned aggregates, Pb2+ cannot induce DNAzyme cleavage and no color change can be observed. (f) For tail-to-tail aligned aggregates, Pb2+ can induce DNAzyme cleavage and color change [26]. (g) A highly active and stable DNAzymenanotube hybrid [29]. The DNAzyme is immobilized on a carbon nanotube and the hybrid can perform efficient cleavage reactions with high turnover numbers. www.sciencedirect.com Current Opinion in Biotechnology 2006, 17:580588

582 Chemical biotechnology

order to assemble nanoparticles, the substrate strand was extended at both ends with segments that were complementary to DNA fragments attached to the nanoparticles. On assembly of the nanoparticles into an aggregate, a red to blue colour change is observed that results from a shift in the surface plasmon resonance properties of the particle. If the nanoparticles were aligned in a head-to-tail manner (Figure 1c), a heating and cooling (annealing) step was needed to form blue-colored aggregates [23]. In the presence of Pb2+, the assembly was inhibited because of substrate cleavage. Changing the alignment to tail-to-tail (Figure 1d) allowed the assembly to occur at ambient temperatures [25]. The reverse process Pb2+induced disassembly of nanoparticle aggregates was also studied [26,28]. Interestingly, if the particles were aligned head-to-tail, the DNAzyme was not active (Figure 1e). Activity and disassembly of nanoparticles were observed for tail-to-tail aligned aggregates (Figure 1f). Because of the accompanying red/blue color transitions, these materials are useful for the colorimetric sensing of Pb2+ or other metal ions [17,23,24,25,26,28]. These research efforts have led to the use of DNAzymes to proofread and correct errors or defects in nanomaterial assembly, an important task for practical applications of nanomaterials [27]. The same Pb2+-specic DNAzyme was also immobilized onto carbon nanotubes in collaborative work between the groups of Lu, Kane and Dordick (Figure 1g) [29]. The immobilized DNAzyme formed a highly stable hybrid with nanotubes and maintained high activity; over 400 turnovers were observed for each molecule of DNAzyme. Such high activity could facilitate many applications, ranging from the directed assembly of nanotubes to nanoscale cellular therapeutics. Along the same lines of surface immobilization, the DNAzyme was covalently attached to gold surfaces [30] and gold-coated nanocapillary membranes. In this case, enzyme activity was maintained even after storage in a dried state for 30 days at room temperature [31]. Micro- and nanouidic devices are promising platforms for several applications, including sensing and drug delivery. The use of DNAzymes in such devices would enable the analysis of extremely small samples, unattended longterm monitoring, and potential multiplex array sensing. The Pb2+-specic DNAzyme discussed above was placed in a microuidic device [32] and less than 1 nL of DNA was found to be needed to monitor the cleavage reaction for Pb2+ sensing. The DNAzyme was also assayed in a nanouidic device containing two perpendicular channels interfaced by a nanocapillary array interconnect. This voltage-controlled device was capable of detecting Pb2+ concentrations as low as 11 nM [33]. Seeman, Ellington and co-workers [34] recently incorporated a Cu2+-dependent DNAzyme [35] with optimized
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cleavage efciency [34] into a two-dimensional array based on the DNA double crossover motif (a rigid DNA building block frequently used in DNA nanotechnology). The array was designed to contain two types of alternating stripes: one from the DNAzyme and the other from an embedded DNA hairpin. The separation between the stripes was 32 nm. After addition of Cu2+, the DNAzyme was cleaved off the array and the separation changed to 64 nm [34]. The authors propose that many DNAzymes responsive to different chemical stimuli could be used to construct such nanostructures the properties of which can then be controlled by those stimuli in a programmable manner.
DNAzyme-based DNA molecular devices

Mao and co-workers [36,37,38] have used DNAzymes to construct molecular motors with open-close motions and walking motions. For example, a DNAzyme (Figure 2a, in green) was anked by two double-stranded overhangs to form a closed structure. The addition of substrate DNA (in purple) forced the device to open. Subsequently, the substrate was cleaved by the DNAzyme into two pieces and released, which moved the device back to the closed state. The device will continue to operate as long as free substrate is present [36]. By introducing a non-cleavable substrate, the motion of the device can be stopped; removal of the non-cleavable substrate can resume the motion [37]. A DNAzyme was also designed to walk along a DNA track (Figure 2b) [38]. Initially, the DNAzyme was hybridized to the substrate DNA, but the hybridization was designed to be asymmetric with one arm being longer than the other; one end of the substrate was hybridized to a DNA track (in blue). Cleavage of the substrate released the shorter substrate fragment leaving the corresponding free DNAzyme portion available to seek a nearby free, full-length substrate. Because the stability of hybridization to the full-length substrate was higher, the DNAzyme moved completely to a new substrate to perform a second cleavage reaction. Up to three migration steps have been demonstrated with the device.

Aptamers in nanotechnology
General considerations

In parallel to protein-based antibodies, aptamers are nucleic-acid-based binding molecules [1214]. Aptamers are obtained by a combinatorial selection method known as systematic evolution of ligands by exponential enrichment (SELEX), in which DNA molecules with the desired binding properties are isolated from a library containing as many as 1015 random sequences. What makes aptamers unique and useful in nanotechnology is the fact that an aptamer can be controlled in three distinct states: free in solution, bound to its target molecule or bound to its complementary DNA [39,40]. Switching between the three states can generate
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Figure 2

DNAzyme-based devices. (a) A nanomotor with open-close motors powered by a DNAzyme [36,37]. When the DNAzyme (in green) is free of substrate, the device is in the closed state. Hybridization to a substrate (in purple) opens the device, which is subsequently closed by DNAzyme cleavage and product release. (b) A DNAzyme nanowalker [38]. A DNAzyme (in green) is initially hybridized to the first substrate (in purple) immobilized on a DNA track (blue). Cleavage of the substrate induces migration of the DNAzyme to a nearby new substrate.

mechanical motions, induce or release constraints, and associate or dissociate chemicals or oligonucleotides. Many recent studies have taken advantage of this feature.
Aptamer-templated material assembly

Aptamers have been recently employed to assemble nanoparticles, and many of the resulting materials are dynamic in nature and sensitive to chemical stimuli. In one such study, Willner and co-workers [41] functionalized gold nanoparticles with an antithrombin aptamer. In the presence of high concentrations of thrombin, the nanoparticle solution showed some turbidity, which was attributed to each thrombin molecule interacting with two thrombin aptamers [42] and initiating nanoparticle assembly to form aggregates (Figure 3a). Similarly, Chang and co-workers employed platelet-derived growth factor (PDGF) to assemble PDGF-aptamer-functionalized gold nanoparticles with a visible red-to-purple color change in the presence of PDGF [43]. As in the DNAzyme studies described earlier, the color change results from changes in the surface plasmon resonance coupling of the nanoparticles on binding. These assemblies are limited to molecules that can bind multiple aptamers. In a complementary study, Liu and Lu [44] reported a more general design in which aptamers were used as a linker to assemble DNA-functionalized gold nanoparticles (Figure 3b). In the presence of target molecules, the aptamer switched its structure and the nanoparticles dissociated. As a result, purple-colored aggregates dispersed into red colored individual nanoparticles. This design can be used to obtain nanomaterials sensitive to a broad range of molecules by simple replacement of the aptamer sequences. Nanostructures sensitive to adenosine, cocaine and potassium ions have been demonwww.sciencedirect.com

strated [44,45]. Because of its generality, more complex systems were built to be responsive to multiple chemical inputs with controllable input cooperativity [45]. For example, the disassembly of materials shown in Figure 3c required the presence of both adenosine and cocaine, whereas the disassembly of materials shown in Figure 3d can happen with either molecule. Besides nanoparticle aggregates, protein arrays have also been made by aptamer-directed assembly. For instance, Yan and co-workers [46] used thrombin aptamers to form one-dimensional thrombin arrays (Figure 3e). Because aptamers are also a fragment of DNA, such a design can be conveniently incorporated into the construction of a DNA scaffold without the need to introduce other functional groups for protein conjugation. Incorporation of a uorophore-modied aptamer into such DNA arrays enabled uorescent detection of thrombin (see also Update). Aptamers have also been applied to control the chemical and physical properties of other materials. Ellington and co-workers [47] employed aptamers to control the emission of semiconductor nanocrystals called quantum dots (QDs) (Figure 3f). A short piece of quencher-labeled DNA was hybridized to a thrombin aptamer attached to a QD; therefore, the QD emission was initially quenched. Addition of thrombin resulted in dissociation of the quencher-labeled DNA and unmasked the QD emission. Willner and co-workers [48] also made use of thrombin aptamers in this case for the immobilization of platinum (Pt) nanoparticles. The authors immobilized thrombin aptamers on a gold surface and functionalized Pt nanoparticles with thrombin aptamers (Figure 3g) [48]. Thrombin was then sandwiched between the two
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Figure 3

Aptamer-templated nanomaterial assembly. (a) The assembly of thrombin-aptamer-functionalized gold nanoparticles by thrombin [41]. Each thrombin molecule (yellow) can bind two aptamers. The same concept has also been used to assemble platelet-derived growth factor (PDGF)-aptamerfunctionalized gold nanoparticles [43]. The nanoparticles are depicted in red or blue; a red to blue/purple color transition is observed on assembly, owing to surface plasmon effects. (b) Aptamer-assembled nanoparticle aggregates sensitive to chemical inputs such as adenosine, cocaine and K+ [44]. Upon binding to adenosine, the nanoparticles are dissociated into the dispersed state. Nanomaterials responsive to adenosine and cocaine with (c) high or (d) no cooperativity [45]. (e) Patterning thrombin arrays using thrombin aptamers as a capturing agent [46]. (f) Aptamer-controlled emission properties of quantum dots (QDs) [47]. A quencher-labeled DNA is positioned close to the QD by hybridization to an attached thrombin aptamer. Addition of thrombin releases the quencher labeled DNA, unmasking the fluorescence. (g) Thrombin-mediated immobilization of Pt nanoparticles on a gold surface for catalyzing the reduction of H2O2 [48].

aptamers and, as a result, the Pt nanoparticle was immobilized. Pt nanoparticles can catalyze the reduction of H2O2 to H2O to generate electrochemical signals. This system is useful for the amplied electrochemical detection of thrombin.
Aptamer-based DNA molecular devices

Most DNA-based molecular devices are powered by the energy generated from DNA hybridization. By contrast, in a biological system, most motors are fueled by ATP hydrolysis. Recent work on aptamer-based devices has shown great promise in mimicking biological systems. The rst example, which can be considered an aptamerbased DNA motor, was reported by Li and Tan [49]. A piece of guanine-rich DNA (Figure 4a, top in green) can
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form a quadruplex by binding to K+, and this piece of DNA can be considered a K+ aptamer. The addition of complementary DNA (in blue) forces the quadruplex to open leaving the DNA in a duplex form. By leaving a tail on the added DNA, when the full complementary DNA (in orange) is added, the K+ aptamer DNA is released to re-associate with K+ and form a quadruplex structure. The aptamer can be switched in the state of binding to K+ or binding to its complementary DNA for over ten cycles. The same sequence can also bind thrombin [50]. Simmel and co-workers [51] have extended the quadruplex region with a tail (Figure 4b, in black) to facilitate the release of thrombin by a complementary strand. As in the earlier study, the aptamer DNA switched between binding to thrombin and to the complementary DNA for
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Functional DNA nanotechnology Lu and Liu 585

Figure 4

Aptamer-based DNA devices. (a) A DNA nanomotor powered by K+ and DNA [49]. The motor (in green) adopts a quadruplex structure by binding K+ (red). In the presence of complementary DNA (blue), the motor becomes part of a DNA duplex. Cycling of the motor is realized by the addition of another DNA (orange) to free the motor. (b) A DNA nanomotor powered by thrombin and DNA [51]. The motor (in green with a black tail) adopts a quadruplex structure by binding thrombin. In the presence of complementary DNA, the motor becomes part of a DNA duplex and leaves the thrombin. Cycling of the motor is realized by the addition of another DNA (in orange) to free the motor. (c) A DNA device powered by adenosine and adenosine deaminase [52]. A short DNA molecule (in blue) is hybridized to an adenosine aptamer (in green). Adenosine can induce structure switching in the aptamer, releasing the short DNA. The device is cycled by adenosine deaminase to turn adenosine into inosine. (d) Inhibition of thrombin (yellow) activity by a covalently linked thrombin aptamer (green) and activity rescue directed by a specific DNA (in orange) [53]. Immobilization on a glass surface allows convenient optimal detection. (e) Control of RNA folding by an aptamer [55]. Two pieces of DNA (in blue) are covalently attached to an RNA molecule (orange). The addition of a hemin aptamer complementary to the two DNA molecules leads to the misfolding of the RNA. Hemin can bind to the aptamer and relieve constraints on the RNA to allow it to fold correctly.

multiple cycles. Although aptamers were involved, these two devices still relied on the energy from DNA hybridization. By introducing a protein enzyme that can chemically modify the target molecule of an aptamer, Nutiu and Li [52] demonstrated a device free of external DNA. Initially, a short DNA strand (Figure 4c, in blue) was hybridized to an adenosine aptamer DNA (in green). In the presence of adenosine, the DNA in blue was released and the aptamer DNA bound two adenosine molecules. The device was cycled by the addition of adenosine deaminase, which transformed adenosine into inosine. As the aptamer cannot bind inosine, it re-associates with the short DNA in blue. Therefore, adenosine was the fuel for the system and inosine was the waste. Thrombin activity can be inhibited upon binding to its aptamer; therefore, controlling aptamer binding to thrombin can be used to regulate thrombin activity. Willner and co-workers [53] inhibited thrombin by covalently attaching thrombin aptamers to the molecule via a singlestranded DNA linker (Figure 4d). The addition of DNA complementary to the linker forced the aptamer to leave thrombin, thus rescuing enzyme activity. In another recent study, Miduturu and Silverman [54,55]
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used chemically controlled transitions between DNA duplexes and aptamertarget complexes to control the folding of macromolecules. Two short DNA molecules were covalently attached to the 160-nucleotide Tetrahymena group I intron P4P6 RNA domain, which has well-characterized folding behaviour (Figure 4e). In the presence of a DNA complementary to the two short DNAs, RNA folding was constrained because the free energy favored formation of DNA duplexes. The complementary DNA also contained a hemin aptamer overhang. In the presence of hemin, the constraint was released and the RNA folded into its native state owing to the release of one DNA duplex. Although performed on an RNA molecule, the same method could be applicable to controlling the folding of other biopolymers and polymers.

Aptazymes in nanotechnology
DNAzymes can perform chemical modications on nucleic acids, while aptamers can bind a broad range of molecules. A combination of the two has generated a new class of functional nucleic acids known as allosteric DNAzymes or aptazymes [56]. Liu and Lu [57] have employed an adenosine-dependent aptazyme [58], built on the basis of the Pb2+-specic DNAzyme, to assemble
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gold nanoparticles. In the presence of adenosine, the substrate was cleaved and the assembly inhibited.

6.

Feldkamp U, Niemeyer CM: Rational design of DNA nanoarchitectures. Angew Chem Int Ed Engl 2006, 45:1856-1876. Simmel FC, Dittmer WU: DNA nanodevices. Small 2005, 1:284. Yan H, Park SH, Finkelstein G, Reif JH, LaBean TH: DNAtemplated self-assembly of protein arrays and highly conductive nanowires. Science 2003, 301:1882-1884. Sharma J, Chhabra R, Liu Y, Ke Y, Yan H: DNA-templated self-assembly of two-dimensional and periodical gold nanoparticle arrays. Angew Chem Int Ed Engl 2006, 45:730-735.

Conclusions
In summary, although their appearance in the literature has been very recent, functional DNA molecules such as aptamers, DNAzymes and aptazymes have already found application in almost every aspect of DNA nanotechnology. Therefore, the eld of DNA nanotechnology has been signicantly broadened, and the resulting new materials and devices can penetrate into many other elds for practical applications, such as sensing, environmental monitoring, medical diagnostics, drug screening, therapeutics, nanoelectronics, nanophotonics, and quantum computing [18,59,60]. In the future, we are likely to see the incorporation of individual devices into more complex multifaceted systems. Multidisciplinary research, such as the application of functional DNAbased nanodevices in microuidics, biophysics and biology, will broaden the practical applications of such devices in many new areas. Importantly, with the power to combinatorially select functional nucleic acids, these molecules will become a rich source for developing nanotechnology.

7. 8.

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Update
Yan and co-workers recently prepared two-dimensional DNA arrays containing uorophore-labeled thrombin aptamers, which showed increased uorescence in the presence of thrombin [61]. Such a design allows the generation of programmable sensor arrays that can be imaged on the surface.

Acknowledgements
Research by the Lu group reviewed in this article is based upon work supported by the Department of Energy through an Environmental Remediation Sciences Program (DE-FG02-01ER63179), the National Science Foundation through a Nanoscale Science and Engineering Center grant (DMR-0117792), the US Army Research Laboratory and the US Army Research Ofce under grant number DAAD19-03-1-0227, and by the Illinois Waste Management Research Center under Contract number HWR04187.

References and recommended reading

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27. Liu J, Wernette DP, Lu Y: Proofreading and error removal  in a nanomaterial assembly. Angew Chem Int Ed Engl 2005, 44:7290-7293. This paper rst proposed and demonstrated the concept of using functional biomolecules not only to template nanomaterial assembly, but also to perform proofreading and error correction functions in a way similar to biological systems. 28. Liu J, Lu Y: Design of asymmetric DNAzymes for dynamic control of nanoparticle aggregation states in response to chemical stimuli. Org Biomol Chem 2006, 4:3435-3441. 29. Yim T-J, Liu J, Lu Y, Kane RS, Dordick JS: Highly active and  stable DNAzyme-carbon nanotube hybrids. J Am Chem Soc 2005, 127:12200-12201. A Pb2+-specic DNAzyme was immobilized onto carbon nanotubes to form stable and highly active hybrids capable of performing over 400 catalytic turnovers. 30. Swearingen CB, Wernette DP, Cropek DM, Lu Y, Sweedler JV, Bohn PW: immobilization of a catalytic DNA molecular beacon on Au for Pb(II) detection. Anal Chem 2005, 77:442-448. 31. Wernette DP, Swearingen CB, Cropek DM, Lu Y, Sweedler JV, Bohn PW: Incorporation of a DNAzyme into Au-coated nanocapillary array membranes with an internal standard for Pb(II) sensing. Analyst 2006, 131:41-47. 32. Shaikh KA, Ryu KS, Goluch ED, Nam J-M, Liu J, Thaxton CS, Chiesl TN, Barron AE, Lu Y, Mirkin CA et al.: A modular microuidic architecture for integrated biochemical analysis. Proc Natl Acad Sci USA 2005, 102:9745-9750. 33. Chang I-H, Tulock JJ, Liu J, Kim W-S, Cannon DM Jr., Lu Y, Bohn PW, Sweedler JV, Cropek DM: Miniaturized lead sensor based on lead-specic DNAzyme in a nanocapillary interconnected microuidic device. Environ Sci Technol 2005, 39:3756-3761. 34. Garibotti AV, Knudsen SM, Ellington AD, Seeman NC: Functional DNAzymes organized into two-dimensional arrays. Nano Lett 2006, 6:1505-1507. 35. Carmi N, Balkhi HR, Breaker RR: Cleaving DNA with DNA. Proc Natl Acad Sci USA 1998, 95:2233-2237. 36. Chen Y, Wang M, Mao C: An autonomous DNA nanomotor powered by a DNA enzyme. Angew Chem Int Ed Engl 2004, 43:3554-3557. 37. Chen Y, Mao C: Putting a brake on an autonomous DNA  nanomotor. J Am Chem Soc 2004, 126:8626-8627. A DNAzyme-based free-running nanomotor was previously reported. This paper demonstrated a way to control the free-running nanomotor by using inactive, but structurally related, DNA sequences. 38. Tian Y, He Y, Chen Y, Yin P, Mao C: A DNAzyme that walks  processively and autonomously along a one-dimensional track. Angew Chem Int Ed Engl 2005, 44:4355-4358. A sophisticated system was designed to transport a DNAzyme along a DNA track. In this walking system, the walker carries an enzymatic function that enables it to move forward. 39. Nutiu R, Li Y: Structure-switching signaling aptamers. J Am Chem Soc 2003, 125:4771-4778. 40. Nutiu R, Li Y: Structure-switching signaling aptamers:  transducing molecular recognition into uorescence signalling. Chem Eur J 2004, 10:1868-1876. Contains a detailed description of how to tune aptamer binding states between the cognate analyte and the complementary DNA. 41. Pavlov V, Xiao Y, Shlyahovsky B, Willner I: Aptamerfunctionalized Au nanoparticles for the amplied optical detection of thrombin. J Am Chem Soc 2004, 126:11768-11769. 42. Padmanabhan K, Padmanabhan KP, Ferrara JD, Sadler JE, Tulinsky A: The structure of a-thrombin inhibited by a 15-mer single-stranded DNA aptamer. J Biol Chem 1993, 268:17651-17654. 43. Huang C-C, Huang Y-F, Cao Z, Tan W, Chang H-T: Aptamer modied gold nanoparticles for colorimetric determination of platelet-derived growth factors and their receptors. Anal Chem 2005, 77:5735-5741. www.sciencedirect.com

Platelet-derived growth factors (PDGF) were used to assemble PDGF aptamer-functionalized gold nanoparticles, taking advantage of the fact that each PDGF can bind to two PDGF aptamers. The assembly process was monitored through the use of a color-change assay. 44. Liu J, Lu Y: Fast colorimetric sensing of adenosine and  cocaine based on a general sensor design involving aptamers and nanoparticles. Angew Chem Int Ed Engl 2006, 45:90-94. Two chemical responsive systems were demonstrated that use aptamer DNA to template the assembly of nanoparticles. In the presence of target molecules, aggregated nanoparticles disassembled into a dispersed state in seconds, accompanied by a purple-to-red color change. Such materials are useful for colorimetric sensing of a broad range of analytes. 45. Liu J, Lu Y: Smart nanomaterials responsive to multiple  chemical stimuli with controllable cooperativity. Adv Mater 2006, 18:1667-1671. Materials whose assembly could be controlled by multiple chemicals were prepared on the basis of aptamer-linked nanoparticle aggregates; the cooperativity between chemical stimuli can be controlled by different material designs. Any two combinations of adenosine, cocaine or potassium ions were demonstrated to control nanoparticle assembly states and optical properties. 46. Liu Y, Lin C, Li H, Yan H: Aptamer-directed self-assembly of  protein arrays on a DNA nanostructure. Angew Chem Int Ed Engl 2005, 44:4333-4338. In this study, a thrombin aptamer was incorporated into a DNA periodic structure. Binding of thrombin to the DNA structure enabled the generation of a thrombin array. 47. Levy M, Cater SF, Ellington AD: Quantum-dot aptamer beacons  for the detection of proteins. ChemBioChem 2005, 6:2163-2166. A quencher-labeled DNA was positioned close to a quantum dot by hybridization to an attached thrombin aptamer. The addition of thrombin released the quencher labeled DNA, unmasking the uorescence. This device has been used for sensing applications. 48. Polsky R, Gill R, Kaganovsky L, Willner I: Nucleic acidfunctionalized Pt nanoparticles: catalytic labels for the amplied electrochemical detection of biomolecules. Anal Chem 2006, 78:2268-2271. 49. Li JJ, Tan W: A single DNA molecule nanomotor. Nano Lett 2002, 2:315-318. 50. Bock LC, Grifn LC, Latham JA, Vermaas EH, Toole JJ: Selection of single-stranded DNA molecules that bind and inhibit human thrombin. Nature 1992, 355:564-566. 51. Dittmer WU, Reuter A, Simmel FC: A DNA-based machine that  can cyclically bind and release thrombin. Angew Chem Int Ed Engl 2004, 43:3550-3553. This paper demonstrated the concept of using aptamer-binding to control the conformation of DNA. Importantly, the aptamer can be used as a nanohand to grab and release proteins. 52. Nutiu R, Li Y: A DNA-protein nanoengine for on-demand  release and precise delivery of molecules. Angew Chem Int Ed Engl 2005, 44:5464-5467. Most previously reported DNA machines require the use of DNA hybridization as an energy source. This paper, however, reports the rst DNA machine to be run with non-DNA fuel. Here, a protein enzyme was employed to chemically modify the target of an aptamer thus controlling aptamer conformation in solution, with the target molecule functioning as fuel for the system. 53. Pavlov V, Shlyahovsky B, Willner I: Fluorescence detection of  DNA by the catalytic activation of an aptamer/thrombin complex. J Am Chem Soc 2005, 127:6522-6523. Thrombin activity was inhibited by a covalently attached DNA aptamer; the activity can be rescued by removal of aptamer binding with complementary DNA. 54. Miduturu CV, Silverman SK: DNA constraints allow rational control of macromolecular conformation. J Am Chem Soc 2005, 127:10144-10145. 55. Miduturu CV, Silverman SK: Modulation of DNA constraints that  control macromolecular folding. Angew Chem Int Ed Engl 2006, 45:1918-1921. Several methods to control the folding of a DNA-modied RNA molecule were demonstrated, including the use of a hemin aptamer to control RNA folding by hemin. Current Opinion in Biotechnology 2006, 17:580588

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56. Soukup GA, Breaker RR: Nucleic acid molecular switches. Trends Biotechnol 1999, 17:469-476. 57. Liu J, Lu Y: Adenosine-dependent assembly of aptazymefunctionalized gold nanoparticles and its application as a colorimetric biosensor. Anal Chem 2004, 76:1627-1632. 58. Wang DY, Lai BHY, Sen D: A general strategy for effectormediated control of RNA-cleaving ribozymes and DNA enzymes. J Mol Biol 2002, 318:33-43.

59. Famulok M: Allosteric aptamers and aptazymes as probes for screening approaches. Curr Opin Mol Ther 2005, 7:137-143. 60. Lee JF, Stovall GM, Ellington AD: Aptamer therapeutics advance. Curr Opin Chem Biol 2006, 10:282-289. 61. Lin C, Katilius E, Liu Y, Zhang J, Yan H: Self-assembled signaling aptamer DNA arrays for protein detection. Angew Chem Int Ed 2006, 45:5296.

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