A Synthetic Chemomechanical Machine Driven by Ligand−ReceptorBondingGabriel J. Lavella,* Amol D. Jadhav, and Michel M. Maharbiz†Department of Electrical Engineering and Computer Science, University of California, Berkeley, California 94720, United States

*S Supporting Information

ABSTRACT: The ability to create synthetic chemomechanical machines with engineered functionality promises largetechnological rewards. However, current efforts in molecular chemistry are restrained by the formidable challenges faced inmolecular structure and function prediction. An alternative approach to engineering machines with tailorable chemomechanicalfunctionality is to design Brownian ratchet devices using molecular assemblies. We demonstrate this through the creation ofautonomous molecular machines that sense, mechanically react, and extract energy from ligand−receptor binding. We present aspecific instantiation, measuring approximately 100 nm in length, which actuates upon detection of a streptavidin ligand.Machines were designed through the tailoring of energy landscapes on 3D DNA origami motifs. We also analyzed the responseover a logarithmic concentration ratio (device:ligand) range from 1:101 to 1:105.

KEYWORDS: Brownian ratchet, chemomechanical, DNA, transducer, autonomous

Brownian ratcheting is a mechanism that can be used tocreate mechanically functional and autonomous molecular

machines. Evidence for this exists in the multitudes ofchemomechanical ratchets in nature and the wide range oftasks they perform.1,2 Well known examples include the actinratchets used in filopodia protrusion3,4 and Lysteria mono-cytogenes locomotion.5,6 They distinguish themselves frommolecular motors, such as myosin and kinesin, which operateon mechanochemical cycles, and acquire energy for work fromthe hydrolysis of ATP.7 Instead, ratchets tend to be noncyclic,obtain energy from polymerization reactions, and achievedirected motion by rectifying random substrate movementswith asymmetries in the energy landscape.8,9 While manynatural ratchets have been studied in the context of theirbiological function, the technological pursuit of syntheticchemomechanical ratchets has garnered little attention. Bycomparison, molecular motors are being heavily pursued withapplications in nanoscale transport in mind.8,10 Severalsynthetic motors have been produced; many are walkingdevices that move along tracks and are fueled in artificialenvironments with supplied DNA strands through hydrol-ysis11,12 or hybridization.13,14

Here we demonstrate a molecular machine based on asynthetic Brownian ratchet architecture that senses, mechan-ically reacts, and extracts energy from a streptavidin ligand. We

achieve this through topology and tailoring of energylandscapes and a cascade of ligand−receptor binding eventsthat serve as the Brownian ratchet polymerization reaction. Thelatter distinguishes our architecture from natural systems, wherethe polymerization species is structural and enables userselection of the chemomechanical functionality through thechoice of the receptor (e.g., antibodies, binding pocket RNA, ordesigned probes). We have previously tested the feasibility ofthis characteristic by using bound surface structures andantibodies.15

The basic architecture of our device consists of a clip shapedbackbone (gray, Figure 1a, state X1) conjugated with receptormolecules (orange, Figure 1a, state X1). Before the introductionof ligand molecules, the ratchet thermally vibrates within apotential well governed by the flexure characteristics of thebackbone and the temperature of the bath (depicted notionallyin Figure 1c, State X1). Here, ΔU represents the change inpotential energy originating from the strain energy in thebackbone and stretched molecular complexes. The x-axis, χ,represents the Brownian coordinate, which we have defined as

Received: July 15, 2012Revised: August 24, 2012Published: August 27, 2012

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the symmetric displacement of the tips from their initialposition; as χ increases, the device is moving toward a closedstate.Bonding of a ligand to one of the receptors from the first pair

(Figure 1a, X2) creates an alternative path in the energylandscape that enables a transition between states X1 and X2

(notation X1−2, Figure 1c) by enabling the formation of asandwich bond (as in Figure 1b, IV). Transitioning to this pathrequires that the random thermal input be capable ofsurmounting the forward energy barrier, U1,for and that abound/unbound receptor configuration exists between oppos-

ing receptors (as in Figure 1b, II). As an aside, bound/unboundreceptor configurations arise when the reaction rate forsandwich bond formation is sufficiently higher than thereaction rate for ligand−receptor binding. The contrarycondition would result in the saturation of the receptors andan inability to ratchet, something that will require further studyfor more complex implementations of this architecture. Uponsandwich bond formation, the potential energy of the systemdecreases, and a new potential well (Figure 1c, X2) isestablished, with the equilibrium position, advanced to χ2,eq,(i.e., in the closing direction; χ increasing) and bound by the

Figure 1. Synthetic Brownian ratchet operation. (a) Schematic of the synthetic Brownian ratchet operation at successive states: X1, before ligandintroduction; X2, first binding event; X3, second binding event; and Xn, the final closed configuration . (b) Equilibrium transition at the level of asingle sandwich bond. Energy extraction occurs between nonequilibrium sandwich bond formation, position III, and relaxation to a new equilibriumstate, IV. (c) Characteristic energy landscape at successive device states, X1−X3.

Figure 2. Device backbone topology and receptor/toehold configuration. (a) Diagram depicting backbone composition and biotin receptorplacement using toehold strands. Along a single B-form helix receptors can be placed with the same orientation every 7 nm. (b) Negative stain TEMimages of single devices, toeholds not visible, scale bar = 50 nm. (c) Negative stain TEM of a device ensemble, scale bar = 100 nm. (d)Characterization of interior angle (right); devices bound with poor orientations (left, I and II) were excluded. (e) Agarose gel characterization of alldevice variants without ligand added. (f) Receptor configurations for devices with 7 and 14 nm receptor/toehold spacing. (g) Receptor/toeholdconfigurations for controls A and B.

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reverse, U2,rev, and forward, U2,for, energy barriers. Figure 1bdepicts this transition X1−2, at the level of an individual receptorpair. Each pair will proceed through a series of events beginningwith ligand binding in state X1 (Figure 1b, II), followed by anonequilibrium sandwich bond formation (Figure 1b, III) andconcluding with relaxation to a new equilibrium (Figure 1b,IV). The power for the transition is extracted from the ligand−receptor binding free energy during the simultaneous relaxationof the backbone and tensioning of the sandwich bond. Thistransfer of free binding energy to mechanical work can beaccounted for with an additional term in the Gibbs free energyequation and has been examined extensively in recentliterature.16−18 In essence, the ratchet physically pulls energyfor mechanical work from the receptor−ligand bond.Sequential transitions such as these, where energy is extractedat each step and new landscapes are established, proceed untilthe ratchet is closed (Figure 1a, Xn); on the energy landscapedepictions, this can be seen as a progression toward a lowerabsolute equilibrium.It is important to note that not all receptor pairs need to

form bonds, ratcheting can proceed by leapfrogging ratchetsteps because of the distribution of random thermalperturbations. Adjusting the spacing of receptors or the rigidityof the backbone can be used to control how often this occurs;this affects the kinetics of ratcheting. Similarly, increasing thebackbone stiffness will present a higher and steeper energybarrier and therefore will reduce the likelihood of a statetransition for a given period of time. Increasing the axial

stiffness of the toeholds has the effect of allowing more energyto be transferred to the backbone and therefore the amount ofwork that can be extracted from the ligand−receptor bond.To test the architecture, we created devices with similar

topologies to those presented in Figure 1 using the honeycombarray DNA origami19,20 method. We designed the backboneusing 10 B-form DNA helices stressed by base pair insertionsand deletions into a symmetrical clip shape (Figure 2a, eachDNA helix is represented as a gray tube). Adjacent helices werejoined by Holliday junction crossovers every 21 bp. We thenmodified the backbone design by eliminating sequences alongthe center helix (green in Figure 2a) and inserting 44 bptoeholds strands modified with biotin at the 5′ end to serve asreceptor molecules. It was possible to place toeholds withidentical orientations every 21 bp or 2 complete turns of thehelix at 10.5 bp per turn. We selected base pair locations alongthe turn such that their orientation was approximatelyperpendicular to the helical axes and in the plane of the firstvibrational mode. Devices were then fabricated through thermalannealing in a one-pot reaction using a 7249 bp single-strandedM13mp18 circular DNA scaffold and a set of complementarystaple and toehold strands. Negative stain transmission electronmicroscopy (TEM) images of single devices (Figure 2b) anddevice ensembles (Figure 2c) show the device state beforeintroduction of the ligand and ratcheting. Detailed descriptionsof the fabrication and imaging techniques can be found in theSupporting Information. Measurements of the backboneinterior angle were taken from TEM images (Figure 2d,

Figure 3. Negative stain TEM and gel electrophoresis analysis of device response. (a) Negative stain TEM images of reaction products: ratcheteddevice (top), open device (bottom, left), and device dimers (bottom, right). (b) Variations in ratcheted closure resulting from different sandwichbond configurations. (c) Gel electrophoresis analysis of ratcheting after a reaction with streptavidin ligand. Device concentration ratios were 1:103

(devices:ligand). (d) Negative stain TEM images of ratcheted devices extracted from band, r1. (e) Normalized intensity plot analysis of gelelectrophoresis. Ratcheted devices appear in band r1 and device dimers appear in band d2. Scale bars = 50 nm and insert scale bar = 10 nm.

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right) by excluding devices that did not bind in the correctorientation for inspection to the TEM grid; we identified thesedevices as either possessing a wide flat backbone (Figure 2d, I)or devices which were badly twisted (Figure 2d, II). Correctlyoriented devices showed no signs of either abnormality (Figure2d, III).We constructed four device variants with different receptor/

toehold configurations; two active device sets, expected toratchet, with receptor spacings of 7 and 14 nm (Figure 2f) andtwo control sets with gaps in receptor molecule placement(Figure 2g), wherein a high forward energy barrier wasintroduced and closing could not occur in the absence ofratcheting phenomena. In control A, we eliminated the middlefive receptors on the top arm and six receptors on the bottomarm. Similarly, in control B we removed the first nine receptorpairs, so that ratcheting would be highly unlikely. DNAsequences and strand layouts for each device are provided inthe Supporting Information.Unlike previous DNA origami devices, gel electrophoresis

(Figure 2e) showed that correctly formed devices appearbehind the large smear of the fastest moving band. We believethis occurs because structures with correctly formed toeholdswithin the clip retard their migration in the gel, while structureswith partially formed toeholds in the interior of the devicetranslate more rapidly. Agarose gel images (1.75% agarose, 4 V/cm) of devices show correctly formed devices in bands at ∼2.2

kbp. Devices with 7 nm toehold spacing moved at a slightlyslower rate as a result of their higher molecular weight and theencumbrance presented by larger number toeholds.Estimates of the energy landscapes were made using a toy

model (Supporting Information). We assessed whethertransitions over the forward energy barriers were both likelyand statistically favored. Further, we ensured that the backbonedesign was rigid enough so that the likelihood of thermalvibrations closing the device without ratcheting was extremelyremote.To inspect the ratcheting phenomena, we first gently

introduced the tetravalent streptavidin ligand at a concentrationration of 1:103 (device:streptavidin) into a solution containingdevices with 7 nm toehold spacings. All ratcheting reactionswere performed at 25 °C for 20 min in a buffer solution (0.5×phosphate buffered saline, 0.5× TBE, pH 7.4). A negative stainTEM, post reaction, shows the formation of three distinctproducts: ratcheted devices, nonratcheted devices withstreptavidin bound, and device dimers (Figure 3a). Thestreptavidin ligand can be clearly seen down the centerline ofthe device, a high-magnification image confirms tetravalentstreptavidin (see Figure 3a, insert). The distance betweenopposing arms, 20 nm, measures the approximate height of thedry form DNA sandwich bond. Higher quantities of boundstreptavidin resulted in conformation changes with a slightlyhigher degree of closing (Figure 3b). We believe this occurs as a

Figure 4. Gel electrophoresis analysis of Brownian ratchet response over a logarithmic concentration ratio gradient. Agarose gel analysis of the deviceresponse over a concentration ratio gradient for the configurations with (a) 7 and (b) 14 nm spacings between receptors. Normalized intensitycomparison across gradient for ratcheted bands r1 and r2 for devices with (c) 7 and (d) 14 nm spacings between receptors. Normalized intensitycomparison across gradient for bands with dimer formation,d2, trimers, d3, and devices that failed to ratchet, d1, with (e) 7 and (f) 14 nm spacingsbetween receptors.

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result of the numerous receptor bonding configurations thatcan arise during ratcheting (see Figure 3b, I−III).In order to confirm ratcheting was responsible for the

conformation change, we performed a gel analysis of thereaction products for the active and control device variants. Forthe devices with 7 and 14 nm toehold spacings, a new forwardband (Figure 3c, r1, ∼470 bp) appears ahead of the originaldevice band. Excision of this band followed by purification andnegative stain TEM shows devices in a closed state (Figure 3d).The unclosed device band remains (∼2.2 kbp, Figure 2d),indicating that many devices did not ratchet at all or that theyratcheted closed and reopened. The trailing and diminishingsmear on the forward band, r1, suggests that a small quantity ofdevices partially ratcheted closed. In the controls ( Figure 2g,controls A and B), no ratcheting occurs as indicated by the lackof the forward band, r1. A normalized gel intensity image(Figure 3e) comparing the device response for the variousconfigurations shows a strong response for the 7 nmconfiguration, a comparatively weaker response for the 14 nmconfiguration, and no ratcheting response for either control Aor B. As expected, the greater receptor spacing presents ahigher forward energy barrier which hinders ratcheting. In alldevice variants the dimer band, d2, can be seen between 3.6 and4.0 kbp (Figures 3c and 2e). Conceivably, the long toeholdaided in the formation of dimers as this extends beyond thecharge repulsion layer. A future challenge will be to assemblestructures that sterically hinder dimerization.We next explored the response of the active device variants

over a logarithmic concentration ratio range from 1:101 to 1:105

(devices:streptavidin). Gel analysis indicated that devices with 7and 14 nm receptor configurations exhibit similar trends(Figure 4a,b). In Figure 4a,b the reference lane, R, contained nostreptavidin. For low-concentration ratios, 1:101 (devices:strep-tavidin), few, if any, devices ratcheted as indicated by therelatively weak fluorescent intensity of the ratchet band, r1(Figure 4c,d). Device dimer formation however is evident butminimal in both active device variants (Figure 4e,f, bands d2and d3). Ratcheting becomes pronounced at concentrationratios greater that 1:102; at concentration ratios greater than1:103, a new ratcheted band, r2, results as the quantity of boundstreptavidin per device slows the device migration in the gel.Interestingly, the aggregate intensity of r1 and r2 remainsrelatively constant through high concentrations; this impliesthat the reaction rate for sandwich bond formation is occurringmuch faster than ligand−receptor bonding from the bulksolution. It is important to highlight that conceivably, in thecontrary condition, we would observe a device that actuateswithin band range of concentration ratios. Dimer, d2, andtrimer, d3, formation peaks at low concentrations then graduallydecreases (Figure 4e,f) at high concentrations where thebinding of free ligand is stochastically favored over interdevicebinding.Because the ratchet architecture relies ultimately on the

arrangement of molecules and their inherent properties, theycan benefit from incremental improvements in our ability tomodel the energy landscapes and spatially control molecularassemblies, which need not be DNA based. Advances intopological control can permit more strategic receptorsconfigurations and better backbone topologies that will allowfor the high force mechanical responses seen in naturalsystems.3

In addition, it is conceivable that devices such as these canoperate cyclically when an environment variable is modulated.

Examples include periodic changes in pH, which can elicit bondformation or decoupling,21 or periodic changes in the freeligand concentrations. In the latter, the presence of higherquantities of free biotin would result in competition for boundstreptavidin, eliciting reopening, while a decrease would elicitclosing when streptavidin is present.Technological adaptations for tailorable chemomechanical

transduction are immediately recognizable when consideringthe transduction can be coupled to technologically usefuloutputs, such as FRET signals for sensing motifs or the releaseof secondary molecules for programmed artificial chemical tochemical transduction. Such systems could then be used toperform chemical logic, artificial signaling between cells, signalamplification, diagnostics, and protein tracking.

■ ASSOCIATED CONTENT*S Supporting InformationFabrication and experimental methods, detailed descriptions ofthe model used to estimate viable Brownian ratchet topologies,strand sequences, and DNA origami layouts. This material isavailable free of charge via the Internet at http://pubs.acs.org.

■ AUTHOR INFORMATIONCorresponding Author*E-mail: glavella@eecs.berkeley.eduNotesThe authors declare no competing financial interest.

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