From genes to machines

From genes to machines

(Parte 1 de 3)

From genes to machines: DNA nanomechanical devices

Nadrian C. Seeman Department of Chemistry, New York University, New York, NY 10003, USA

The structural properties that enable DNA to serve so effectively as genetic material can also be used for other purposes. The complementarity that leads to the pairing of the strands of the DNA double helix can be exploited to assemble more complex motifs, based on branched structures. These structures have been used as the basis of larger 2D and 3D constructions. In addition, they have been used to make nanomechanical devices. These devices range from DNA-based shape-shifting structures to gears and walkers, a DNA-stress gauge and even a translation device. The devices are activated by mechanisms as diverse as small molecules, proteins and, most intriguingly, other molecules of DNA.

Introduction The half-century since the publication of the Watson– Crick model for DNA structure has seen ramifications of this advance in areas as diverse as medicine, forensics and basic biology, culminating in the sequencing of the human genome. The use of the DNA structure has largely been as a paradigm in understanding fundamental processes in genetics and exploiting that understanding in practical applications. However, another activity involving DNA has been growing for O20 years, based on the idea that objects, arrays and devices could be made from DNA molecules. This effort started from the recognition that stable branched DNA motifs could be made from synthetic strands of DNA with carefully designed sequences, and that these motifs could be made to assemble via stickyended cohesion [1]. The effort to exploit this approach to build matter with nanoscale features and details is termed ‘structural DNA nanotechnology’. It is useful to recall that the DNA double helix is a nanoscale object, with a diameter of w2 nm and a double helical repeat of w3.5 nm.

The initial endeavors in structural DNA nanotechnology were directed at constructing DNA objects, such as a cube [2] or a truncated octahedron [3]. Once suitably rigid motifs were developed, it was possible to build 2D arrays with programmable features [4–10]. The original impetus for building arrays came from the hope of improving macromolecular crystallization [1], but early goals also included organizing nanoelectronics [1] and DNA-based computation [12]. Erik Winfree’s observation that branched DNA molecules with sticky ends could provide the means for implementing computation by Wang tiles

(see Glossary) on the molecular level spurred a lot of activity in the algorithmic assembly of branched nucleic acid molecules [12]. (For further discussion of DNA objects and arrays see Refs [13,14].)

DNA polyhedra and arrays are static objects that represent an approach to controlling nanoscale structure. However, a key aspect of controlling the structure of matter is the ability to make it change its shape. In principle, objects that change their shapes in response to an external stimulus are capable of functional utility, that is, they can function as machines. Given the ease with which it is possible to control the structures of DNA nanoconstructs, it makes sense to see if it is possible to get them to do some work. The past six years have seen an explosion of activity in the area of nucleic-acid-based nanomachines. They work on several different principles, and their movements have been demonstrated using a variety of physical techniques. Here, discussions have been restricted to systems that involve nanomechanical motion, and exclude molecular beacons [15,16]; the article is structured around different types of systems: the exploitation of DNA structural transitions, Watson– Crick and non-Watson–Crick DNA hybridization, protein binding and autonomous devices.

Glossary

Auto-footprinting: The comparison of the hydroxyl radical attack pattern for a strand in a non-standard DNA motif with the pattern when the same strand is paired with its traditional Watson–Crick complement. B-DNA: The standard double-helical structure for DNA in aqueous solution. Double-crossover (DX) motif: The DX motif consists of two DNA double helices linked in two different places. These molecules are related to intermediates in genetic recombination but, in the molecules used in structural DNA nanotechnology, the linkages are between strands of opposite polarity, rather than the same polarity. G-quartet motif: A four-stranded coaxial DNA complex consisting entirely of guanines. The two protons donated by guanine in the Watson–Crick G-C base pair (attached to N and N ) are donated to two acceptors on the major groove side of guanine (O and N ) Paranemic-crossover DNA: A DNA motif that can be formed by reciprocal exchange between strands of the same polarity on two DNA double helices at every possible position. Set strands: DNA strands that bind to a DNA nanodevice to set its structural state. Unset strands: DNA strands complementary to unset strands that remove the set strands from the nanodevice, freeing it up to be set in a new structural state. Wang tiles: A theoretical tool in tiling theory and computation theory. They are tiles with colored edges that assemble into a mosaic according to the local rule that all edges in the mosaic are flanked by the same color. It has been shown that such a form of self-assembly emulates a Turing machine, a generalpurpose computer. Z-DNA: A left-handed form of DNA that can form from conventional DNA molecules if they have a propitious sequence and are in the right environment.Corresponding author: Seeman, N.C. (ned.seeman@nyu.edu).

Review TRENDS in Biochemical Sciences Vol.30 No.3 March 2005 w.sciencedirect.com 0968-0004/$ - see front matter Q 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.tibs.2005.01.007

DNA devices involving DNA structural transitions The first nanomechanical device used to control DNA structure was a successful, but cumbersome, effort to change theposition ofthebranch point ofaDNAcruciform [17]. As shown in Figure 1a, the system consists of a DNA circle that contains a cruciform with four central base pairs that are capable of branch migration. The device exploits the fact that a cruciform will be extruded under conditions of negative supercoiling, and that it will be re-absorbed when the circle is relaxed. The addition and removal of ethidium was used to demonstrate direct control of double-stranded-DNA branch migration, but this system was cumbersome, its structure was not well defined and its scale was huge. The action of the device was demonstrated by restriction combined with hydroxyl radical auto-footprinting. Christof Niemeyer and colleagues have reported another DNA system on this large scale that is based on a structural transition between two states in superhelical DNA [18]. The transition is induced by increasing the concentration of Mg2C, and is demonstrated convincingly by atomic force microscopy (AFM).

The first device that had a well-defined structure was based on the transition from right-handed B-DNA to lefthanded Z-DNA [19]. Although originally conceived in the 1980s, the robust structural basis for demonstrating this device awaited the advent of rigid structural motifs, such as the double-crossover (DX) motif [20].T his B/Z- transition-based device consists of two DX molecules linked by a shaft of double-helical DNA (Figure 1b). The B/Z transition has two requirements: (i) sequences prone to form Z-DNA [typically (CG)n] and (i) solution conditions that promote the transition [21]; the sequence requirement enables control of the transition in space, that is, how much of the DNAwill undergo the transition, and the need for Z-promoting conditions permits control in time. Twenty nucleotide pairs on the connecting shaft fulfill the sequence requirement and they form Z-DNA in the presence of Co(NH3)6 3C. Although quite successful, this system lacks one of the key features desirable in a DNA-based nanomechanical device: (within the limits of some chemical nuance [2]) only two mechanical states are available to any combination of devices because the state of the device is controlled by the addition of a small molecule to the solution. However, this problem was soon solved by nucleic acid hybridization.

Devices controlled by nucleic acid hybridization The key reason to use DNA-based devices is to take advantage of the sequence specificity associated with DNA hybridization. This approach to establishing the states of DNA devices leads to all of the diversity associated with different DNA sequences. Thus, sequence-dependent devices open a vast array of opportunities for simultaneous control in the same environment, perhaps in the same construct. The first hybridization-based device was the DNA tweezers built by Bernard Yurke and colleagues [23]. Subsequently, the method of controlling hybridization introduced by these workers has been used in all other DNA-duplex-based devices. The idea is simple: first, the DNA construct is put into a particular state using a ‘set’ strand that contains a ‘toehold’ on one end that makes it eight bases longer than necessary to pair with the rest of the motif; second, the set strand is removed by using an ‘unset’ strand that is complementary to the entire length of the set strand, thus, leaving the motif free to pair with another set strand in the next cycle of operation. The unset strand is a better pairing partner for the set strand than the motif because there are more base pairs between it and the set strand than there are between the motif and the set strand. The toehold dangles freely in solution when the set strand is paired with the motif. However, when the unset strand is added to the solution, it serves as an initiation point for it to remove the set strand; once bound to the set strand, the unset strand can invade the motif via single-stranded branch migration until the set strand is completely removed. The unset strand can be thought of as the fuel that runs the device and the set strand–unset strand duplex as the waste generated by the device. The efficacy of the tweezers was demonstrated by FRET.

This initial device designed by Yurke et al. [23] lacked robustness because dimers sometimes formed between machine cycles, although later variants from the same group – including an actuator [24] and a three-state device [25] – did not suffer this problem. The first robust device based on hybridization topology was the PX–JX2 device, where PX is paranemic-crossover DNA and JX2 is its topoisomer, one end of which is rotated relative to the

Add intercalator to relax circle

Remove intercalator to stress circle

(b)

Figure 1. Early DNA nanomechanical devices. (a) A device based on DNA supercoiling. The system consists of a small DNA circle that contains a permanent cruciform. The four nucleotide pairs at the base of the cruciform are capable of branch migrationbecause theyarethesame inboth armsofthe extrudedcruciform (left). When the circle is relaxed by the addition of an intercalator, the mobile nucleotide pairs move to the circle. (b) A device based on the B/Z transition of DNA. Two DX molecules (red and blue) are connected by a double-helical-DNA shaft that contains 20 nucleotide pairs (yellow) that are capable of undergoing the B/Z transition. In the B-state, both domains are on the same side of the shaft (top), but when Co(NH ) is added to the solution, the system switches to the Z-state, and the domains are on opposite sides of the shaft. The red and green circles represent a pair of dyes that are used in the FRET experiment that reports their separation: when the solution is in B-promoting conditions, the dyes are close together, but in Z-promoting conditions, they are further apart. Part (b) reproduced, with permission, from Ref. [19] (w.nature.com).

Review TRENDS in Biochemical Sciences Vol.30 No.3 March 2005120 w.sciencedirect.com

other by 1808 [26]. The PX–JX2 device uses two set strands to establish the state of the device, either in the PX con- formation or the rotated JX2 conformation. The machine cycle of this device is shown in Figure 2a.

The robustness and effectiveness of this device were established by gel electrophoresis, but the most convincing evidence for its operation derived from AFM data. A series of DNA trapezoids, formed from three edge-sharing DNA triangles [27], are connected by the device (Figure 2b). In the PX state, the trapezoids are all parallel to each other, whereas in the JX2 state, successive trapezoids have opposite orientation. Consequently, the two states are readily differentiated by observation in the atomic force microscope. Figure 2c illustrates AFM images of these arrays in each state, showing clearly that the device is able to effect these conversions.

The strength of hybridization-based DNA nanomachines is that devices that respond to different DNA signals can be designed easily just by changing the sequence in the region to which the set strands bind. An example of this approach is described in Box 1.

There have been a many devices based on the Yurke et al. [23] strategy. Prominent among them are walking devices [28,29], wherein a walker ‘nanorobot’ takes steps on a ‘sidewalk’ (Figure 3a). Walking devices afford motion between two different molecular species, enabling their relative spatial arrangement to be altered. Ultimately, they will be used to move other molecular species as cargoes and to intertwine polymers in braided configurations. The steps of this walking device were followed by crosslinking aliquots of the system with psoralen [28], but

FRET has also been used for this purpose [29]. Tian and Mao have used this same approach to make DNA-based gears [30]: they demonstrate, using non-denaturing gel electrophoresis, that removal and addition ofstrands totwo linked circular molecules leads to their mutual rotation.

An exciting combination of 2D DNA crystals and hybridization-based nanodevices has been developed by Hao Yan and colleagues [31]. They have inserted a nanodevice into a DNA parallelogram array [6] so that the dimensions of the crystalline repeat can be changed by operating the device (Figure 3b). A stem–loop in one of the parallelogram edges is stabilized by a removable strand that serves as the complement only to its base. When that strand is removed and replaced by a strand that is complementary to the entire stem–loop, the length of the edge is increased. Figure 3b illustrates this notion schematically; in addition, elegant AFM images that demonstrate the functioning of the system are shown. It is worth noting that this is a purely translational motion.

Non-Watson–Crick base-paired motifs The double helix is the most prominent of the unbranched DNA structures, but it is not the only one. The G-quartet motif [32] has been used by several investigators as a component of a DNA-based device. The simplest of these devices, from the laboratories of Weihong Tan [3] and Jean-Louis Mergny [34] are simple shape-shifting systems wherein a single-stranded G-quartet structure is eliminated by the addition of a molecule complementary to the strand; G-quartet-based devices are based on the notion that G-quartet-containing molecules are less stable than

PX JX2

JX2 JX2 JX2

JX2 200 x 200 nm

200 x 200 nm

(c)(a) (b)

Figure 2. The PX–JX device and its applications. (a) The machine cycle of the PX–JX device. The structure of the PX DNA motif (left), wherein two double helices exchange strands of the same polarity at every possible position [51], is shown. The conformation of the motif is established by two set strands – shown in green for the PX state and pink for the JX state. The set strands (green) are removed from the PX molecule by the addition of biotinylated (black dots) unset strands, which can then be removed with magnetic streptavidin beads (1). The resulting naked frame (top; for clarity, the strands of the naked frame are shown in red and blue) can be bound by set strands (pink) to convert the device to the JX state (2). Note that, because the JX conformation lacks two of the crossovers present in the PX state, the lower region of the JX molecule (labeled D and C) is rotated by a half-turn. Steps (3) and (4) restore the PX state. (b) A system to demonstrate the motion of the PX–JX device. The device connects DNA trapezoids (three fused DNA triangles). When the system is in the PX state, all trapezoids point in the same direction, but they point in opposite directions in the JX state. (c) AFM images of the molecules in (b). The PX and the JX strings show the images expected from the schematics in (b). Part (c) reproduced, with permission, from Ref. [26] (w.nature.com).

Review TRENDS in Biochemical Sciences Vol.30 No.3 March 2005 121 w.sciencedirect.com

the same strands bound to their Watson–Crick complements. This approach has been developed further by FriedrichSimmelandcolleaguesintoareversiblethrombinbinding device [35]. A G-quartet-containing DNA aptamer can be released from thrombin by binding a molecule that is largely complementary to the G-quartet-forming region. However, the resulting duplex contains a toehold so that the complementary strand can be removed, leaving the original aptamer strand free to bind to thrombin again. In another approach to using G-quartets, Dipankar Sen and his colleagues employed Sr2C as an agent to produce a ‘pinched duplex’ within a conventional double-stranded molecule containing an interrupted strand of Gs [36]. The oligo-C-based I-motifhas beenused byDongsheng Liu and Mohan Balasubramanian to construct another shapeshifting device [37]. This motif is dependent on the hemiprotonation of cytosine, so the states of the device can be controlled by protonation.

Another device based on non-Watson–Crick base pairing was developed by Chengde Mao and his colleagues [38]. This is based on the reversible formation of a DNA triple helix based on an oligo–purine–pyrimidine sequence. The T-A-T triple helix forms readily at neutral pH on the major groove (Hoogsteen) face of adenine; by contrast, the C-G-C triple helix requires the protonation of cytosine in the major groove, so it only forms at acidic pH. By varying the pH, an ordered triple helix can be formed from a duplex and a disordered strand segment, resulting in a nanomechanical motion that can be monitored by FRET. The change in structure caused by the transition has been used to control chemical reactivity [39].

A DNA-based stress gauge for proteins The machines already described do not work against a specific load, except for those forces in solution that oppose their motion. A DNA-based molecular device has been developed to establish how much work a DNA-distorting protein can do when it binds to DNA [40]. It was prototyped using integration host factor (IHF), a device that bends DNA by 1608 [41] (Figure 4a). The device is similar to the B-Z device, except that it contains three-domain planar motifs, termed TX [7]. The top shaft, which connects the two TX portions of the device, contains an IHF-binding site. In addition, the bottom domain is connected by a pair of sticky ends. For IHF to bind, the sticky ends must be disrupted (Figure 4a). The bending is reported using a pair of dyes, via a FRET measurement. By increasing the strength of the sticky end, one can arrive at a point where the ability to do work is less than that necessary to break the cohesion of the sticky ends. The free energy associated with each sticky end can be calculated from data obtained by John SantaLucia’s laboratory [42].

Autonomous devices All of the devices described are clocked devices: the experimenter changes something about the environment of the device and a structural feature of the device itself is altered. However, it is evident that it would be desirable to create machines that run without experimenter intervention. This has now been achieved in a few instances. In one case, Mao and colleagues built a device based on a DNAzyme that cleaves RNA [43,4]. The advanced

Box 1. A translation device based on the PX–JX2 device

Figure I shows an example of system with the essential features of a DNA-based nanomechanical translation device [52]. This prototype apparatus consists of a DNA diamond (two fused DNA triangles) and a pair of double diamonds connected by two different PX–JX2 devices (see Figure 2 in main text). The orientations of the double diamonds are determined by the set strands. When both devices are in the PX state, the red diamonds are on the top of the device and the blue ones are on the bottom.

Inthe example shown, device 1 (left) is in the PX state, so that the red half of the central double diamond is on the same side as the single diamond; however, device 2 (right) is in the JX2 state, so that the blue half of the right double diamond is on this side. The diamonds all contain sticky ends that are numbered 1–7. As a consequence of the state of the machine, two double-crossover (DX)-motif-containing molecules, the sticky ends of which complement these numbered sticky ends, are selected to bind the device from a group of six in solution; DX2 and DX5 have been selected in the example shown. These DX molecules are ligated to each other and to an initiator DX (not shown), and the continuous strand that results is sequenced. For all four possible states (PX1, PX2; PX1, JX22; JX21, PX2; JX21, JX22), the correct molecule results. This is a translational device because the coding between the sequences of the set strands and the final product is arbitrary.

PX1

46 DX4

56 DX6

DX2 DX5

Figure I. An illustration of the essential features of a translation device based on the PX–JX system. There is no transcriptional relationship between the set strands in the devices and the sequences in the product.

Review TRENDS in Biochemical Sciences Vol.30 No.3 March 2005122 w.sciencedirect.com

version of this device is shown in Figure 4b. The device binds a strand of RNA and it opens. When the RNA is cleaved, the two cleavage products are sufficiently short that they dissociate from the device; when the products dissociate, the device closes. If there is another molecule of the substrate in the solution, it too can be bound and the system can go through another round. The added level of sophistication of this device is that a DNA brake that cannot be cleaved by the DNAzyme can be added to the solution. The machine can be re-activated by removing the brake, using the standard Yurke et al. [23] approach.

John Reif, Andrew Turberfield and colleagues have demonstrated an autonomous walking device that is based on alternating cycles of ligation, followed by cleavage using type-IIS restriction enzymes, followed by ligation [45]. The ends generated by the restriction enzyme are ligated to form a substrate for a different enzyme. This strategy is closely related to the non-mechanical finite-state machines based on linear DNA and type-IIS restriction enzymes developed by Ehud Shapiro and his colleagues (see, for example, Ref. [46]). A cleavage-free strategy for free-running devices has been described by Turberfield et al. [47]. It entails the invasion of loops by strands that are only barely able to do so in the absence of other strands that are ‘catalytic’ in freeing up the loops for hybridization. A cascade approach (called hybridization chain reaction), designed for sensors more than for devices, has been reported recently by Niles Pierce [48]; once begun, a system forms a long double helix based on preferential pairing.

Transcriptional control of nucleic acid devices sits between autonomous devices and clocked devices. In an exciting proof of principle, Wendy Dittmer and Simmel have used transcription of a designed sequence [49] to control the Yurke et al. [23] tweezers. They only close the tweezers with the RNA molecule that is transcribed, and

Set 1ASet 1ASet 2BSet 1ASet 2B

(Parte 1 de 3)

Comentários