The simplest active DNA nanostructures are switches or actuators that can be driven between two conformations. Motion is induced by changes in temperature or ionic conditions, or by the binding of a signalling molecule, often a DNA strand.
Conformation changes induced by changes in environment
Rotary motion can be produced by changing the twist of DNA. Double-stranded DNA with the sequence (CG)n can be flipped from the usual right-handed helix (B-DNA) to a left-handed conformation (Z-DNA). This transition is favoured by high salt concentrations and low temperatures13. One of the earliest nanomechanical DNA devices14 used this transition to change the angle between two rigid DNA tiles connected by a (CG)10 stem. Each tile carried a reporter fluorophore. Förster resonant energy transfer (FRET) between fluorophores allows sensitive measurement of their separation on a nanometre length scale: the efficiency of energy transfer, mediated by a dipole - dipole interaction, scales as the inverse sixth power of their separation15. When the B - Z transition was induced by an increase in ionic strength, FRET measurements showed an increase in the separation between the fluorophores consistent with the expected relative rotation of the tiles by 3.5 turns.
Yang and co-workers converted changes in the twist of DNA into linear motion16. Their device consisted of a closed loop of double-stranded DNA attached to opposite arms of a four-arm Holliday junction (Box 1)17. A Holliday junction can migrate (isomerize) by breaking identical base pairs in one pair of opposite arms and remaking them in the other pair. A change in the conformation of the DNA within the loop was initiated by adding ethidium bromide: this intercalating dye binds between adjacent base pairs, lengthening and partially unwinding the double helix. The resulting stress was relieved by junction migration: by shortening the protruding arms of the junction the loop was allowed to lengthen without changing the total number of twists within it.
Environmentally driven changes in the conformation of single-stranded DNA can induce linear motion. Under slightly acidic conditions, a single strand with appropriately spaced cytosine bases folds into an i-motif " a compact three-dimensional structure that is held together by cytosine-protonated-cytosine base pairs18. In the presence of a near-complementary strand of DNA there is competition between the i-motif and an extended double helix formed by hybridization of the two strands (note that a perfectly complementary strand would itself fold into a stable structure called the G-quadruplex19). The i-motif device can be switched between compact and extended states by changing the pH20, 21. Cyclic switching can be driven by an oscillating chemical reaction22, 23.
The i-motif-to-duplex transition has been made to do mechanical work. One surface of a silicon cantilever was coated with tethered cytosine-containing strands and formation of the i-motif induced a compressive surface stress that bent the cantilever24. The origin of the surface stress, which is also observed when complementary strands hybridize to tethered probes25, is not understood. Electrostatic repulsion between the compact i-motifs plays a part, but the effect persists with high salt concentrations at which interactions over a distance comparable to the separation between strands are effectively screened.
It has also been suggested that the conformational change resulting from pH-dependent binding of a single strand of DNA to a duplex to form a triple-helical structure could be used as the basis of a nanomechanical actuator26, 27.
Devices such as those described above can be used to monitor and report on their environment. Box 2 describes how active DNA nanostructures are being developed as sensors, how elementary logical operations can be performed on their outputs, and how the combination of sensors and computation might be used to create smart drug-delivery systems.
Conformation changes induced by signalling
Yurke and co-workers28 constructed a pair of DNA tweezers with two rigid double-stranded arms connected at one end by a flexible single-stranded hinge (Fig. 2). In the open configuration, the arms rotate freely about the hinge. Single-stranded tails extend from the free end of both arms and serve as attachment points for a control strand — the 'set' or 'fuel' strand — that can pull the arms together by hybridizing to both of them. A short region of the set strand remains single-stranded even when hybridized to the device: it serves as a toehold for hybridization of a complementary 'unset' or 'antifuel' strand that strips the set strand from the device by branch migration (Box 1). Displacement of the set strand generates a double-stranded waste product and resets the device to its initial open configuration. The device can be driven through many cycles of operation simply by repeated sequential addition of set and unset strands. The time to half completion of a single switching operation is 10 s at typical (micromolar) control-strand concentrations: the rate constant for toehold-mediated strand exchange is 105 M-1 s-1 (ref. 29). Operation of the DNA tweezers has recently been characterized using single molecule FRET measurements30.
Figure 2: A DNA nanomachine driven by repeated sequential addition of DNA control strands.
DNA tweezers28 have two double-stranded arms connected by a flexible single-stranded hinge. The 'set' strand pulls the arms into a closed conformation by hybridizing to single-stranded tails at the ends of the arms. A short region of the set strand remains single-stranded even when it is hybridized to the tweezers: this region serves as a toehold that allows the 'unset' strand to hybridize to the set strand and strip it from the device, returning the tweezers to the open configuration and generating a double-stranded waste product. The state of the device can be determined by measuring the separation between donor and acceptor fluorophores (represented by the green triangle and red circle) using FRET.
Strand displacement has been used to effect conformation changes in a wide variety of systems. A number of variations on the tweezers have been reported including a device where the arms are pushed apart instead of being pulled together31 and a three-state device32. Yan and co-workers33 constructed a linear array of rigid DNA tiles in which adjacent tiles could be flipped between cis and trans conformations by stripping away and replacing control strands. Different conformations were easily distinguished by atomic force microscopy. The same device has recently been incorporated into a two-dimensional DNA array34. Feng et al.35 created a two-dimensional array that could be switched between states with different lattice spacings and Hazarika and co-workers36 used strand displacement to reverse the aggregation of gold nanoparticles held together by DNA bridges37. A number of groups have used DNA control strands to switch a section of DNA between a single-stranded state, designed to fold into a compact G-quadruplex stabilized by hydrogen-bonded tetrads of guanine19, and an extended duplex38, 39, 40.
Motion of DNA devices can be triggered using RNA control strands41. By using a specific messenger RNA (mRNA) as a control strand, a tweezer device has been used to sense in vitro transcription42, 43. These experiments show how DNA devices could be controlled by transcriptional circuits44, 45, 46, 47, and point to future applications in which the presence of a specific mRNA within a cell might trigger an event such as the release of a caged drug48.
The maximum force exerted by hybridization of a signalling strand of DNA can be estimated as the free energy change of hybridization divided by the distance through which the hybridizing strands must move together, and is of the order of 10 pN (ref. 28). The fact that protein motors generate similar forces49, 50, 51, 52 encourages speculation that DNA hybridization could be used to drive DNA devices that mimic natural molecular motors.
Strand displacement has been used to effect conformation changes in a wide variety of systems. A number of variations on the tweezers have been reported including a device where the arms are pushed apart instead of being pulled together31 and a three-state device32. Yan and co-workers33 constructed a linear array of rigid DNA tiles in which adjacent tiles could be flipped between cis and trans conformations by stripping away and replacing control strands. Different conformations were easily distinguished by atomic force microscopy. The same device has recently been incorporated into a two-dimensional DNA array34. Feng et al.35 created a two-dimensional array that could be switched between states with different lattice spacings and Hazarika and co-workers36 used strand displacement to reverse the aggregation of gold nanoparticles held together by DNA bridges37. A number of groups have used DNA control strands to switch a section of DNA between a single-stranded state, designed to fold into a compact G-quadruplex stabilized by hydrogen-bonded tetrads of guanine19, and an extended duplex38, 39, 40.
Motion of DNA devices can be triggered using RNA control strands41. By using a specific messenger RNA (mRNA) as a control strand, a tweezer device has been used to sense in vitro transcription42, 43. These experiments show how DNA devices could be controlled by transcriptional circuits44, 45, 46, 47, and point to future applications in which the presence of a specific mRNA within a cell might trigger an event such as the release of a caged drug48.
The maximum force exerted by hybridization of a signalling strand of DNA can be estimated as the free energy change of hybridization divided by the distance through which the hybridizing strands must move together, and is of the order of 10 pN (ref. 28). The fact that protein motors generate similar forces49, 50, 51, 52 encourages speculation that DNA hybridization could be used to drive DNA devices that mimic natural molecular motors.
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