From Brownian to Deterministic Motor Movement in a DNA-Based Molecular Rotor.

Molecular devices that have an anisotropic periodic potential landscape can be operated as Brownian motors. When the potential landscape is cyclically switched with an external force, such devices can harness random Brownian fluctuations to generate a directed motion. Recently, directed Brownian motor-like rotatory movement was demonstrated with an electrically switched DNA origami rotor with designed ratchet-like obstacles. Here, we demonstrate that the intrinsic anisotropy of DNA origami rotors is also sufficient to result in motor movement. We show that for low amplitudes of an external switching field, such devices operate as Brownian motors, while at higher amplitudes, they behave deterministically as overdamped electrical motors. We characterize the amplitude and frequency dependence of the movements, showing that after an initial steep rise, the angular speed peaks and drops for excessive driving amplitudes and frequencies. The rotor movement can be well described by a simple stochastic model of the system.

Electrokinetic Torque Generation by DNA Nanorobotic Arms Studied via Single-Molecule Fluctuation Analysis.

DNA nanotechnology has enabled the creation of supramolecular machines, whose shape and function are inspired from traditional mechanical engineering as well as from biological examples. As DNA inherently is a highly charged biopolymer, the external application of electric fields provides a versatile, computer-programmable way to control the movement of DNA-based machines. However, the details of the electrohydrodynamic interactions underlying the electrical manipulation of these machines are complex, as the influence of their intrinsic charge, the surrounding cloud of counterions, and the effect of electrokinetic fluid flow have to be taken into account. In this work, we identify the relevant effects involved in this actuation mechanism by determining the electric response of an established DNA-based nanorobotic arm to varying design and operation parameters. Borrowing an approach from single-molecule biophysics, we determined the electrical torque exerted on the nanorobotic arms by analyzing their thermal fluctuations when oriented in an electric field. We analyze the influence of various experimental and design parameters on the "actuatability" of the nanostructures and optimize the generated torque according to these parameters. Our findings give insight into the physical processes involved in the actuation mechanism and provide general guidelines that aid in designing and efficiently operating electrically driven nanorobotic devices made from DNA.

Electrical Actuation of DNA-Based Nanomechanical Systems.

DNA nanotechnology provides efficient methods for the sequence-programmable construction of mechanical devices with nanoscale dimensions. The resulting nanomachines could serve as tools for the manipulation of macromolecules with similar functionalities as mechanical tools and machinery in the macroscopic world. In order to drive and control these machines and to perform specific tasks, a fast, reliable, and repeatable actuation mechanism is required that can work against external loads. Here we describe a highly effective method for actuating DNA structures using externally applied electric fields. To this end, electric fields are generated with controllable direction and amplitude inside a miniature electrophoresis device integrated with an epifluorescence microscope. With this setup, DNA-based nanoelectromechanical devices can be precisely controlled. As an example, we demonstrate how a DNA-based nanorobotic system can be used to dynamically position molecules on a molecular platform with high speeds and accuracy. The microscopy setup also described here allows simultaneous monitoring of a large number of nanorobotic arms in real time and at the single nanomachine level.

Energy landscapes of rotary DNA origami devices determined by fluorescence particle tracking.

Biomolecular nanomechanical devices are of great interest as tools for the processing and manipulation of molecules, thereby mimicking the function of nature's enzymes. DNA nanotechnology provides the capability to build molecular analogs of mechanical machine elements such as joints and hinges via sequence-programmable self-assembly, which are otherwise known from traditional mechanical engineering. Relative to their size, these molecular machine elements typically do not reach the same relative precision and reproducibility that we know from their macroscopic counterparts; however, as they are scaled down to molecular sizes, physical effects typically not considered by mechanical engineers such as Brownian motion, intramolecular forces, and the molecular roughness of the devices begin to dominate their behavior. In order to investigate the effect of different design choices on the roughness of the mechanical energy landscapes of DNA nanodevices in greater detail, we here study an exemplary DNA origami-based structure, a modularly designed rotor-stator arrangement, which resembles a rotatable nanorobotic arm. Using fluorescence tracking microscopy, we follow the motion of individual rotors and record their corresponding energy landscapes. We then utilize the modular construction of the device to exchange its constituent parts individually and systematically test the effect of different design variants on the movement patterns. This allows us to identify the design parameters that most strongly affect the shape of the energy landscapes of the systems. Taking into account these insights, we are able to create devices with significantly flatter energy landscapes, which translates to mechanical nanodevices with improved performance and behaviors more closely resembling those of their macroscopic counterparts.

A DNA origami rotary ratchet motor.

To impart directionality to the motions of a molecular mechanism, one must overcome the random thermal forces that are ubiquitous on such small scales and in liquid solution at ambient temperature. In equilibrium without energy supply, directional motion cannot be sustained without violating the laws of thermodynamics. Under conditions away from thermodynamic equilibrium, directional motion may be achieved within the framework of Brownian ratchets, which are diffusive mechanisms that have broken inversion symmetry1-5. Ratcheting is thought to underpin the function of many natural biological motors, such as the F1F0-ATPase6-8, and it has been demonstrated experimentally in synthetic microscale systems (for example, to our knowledge, first in ref. 3) and also in artificial molecular motors created by organic chemical synthesis9-12. DNA nanotechnology13 has yielded a variety of nanoscale mechanisms, including pivots, hinges, crank sliders and rotary systems14-17, which can adopt different configurations, for example, triggered by strand-displacement reactions18,19 or by changing environmental parameters such as pH, ionic strength, temperature, external fields and by coupling their motions to those of natural motor proteins20-26. This previous work and considering low-Reynolds-number dynamics and inherent stochasticity27,28 led us to develop a nanoscale rotary motor built from DNA origami that is driven by ratcheting and whose mechanical capabilities approach those of biological motors such as F1F0-ATPase.

A synthetic tubular molecular transport system.

Creating artificial macromolecular transport systems that can support the movement of molecules along defined routes is a key goal of nanotechnology. Here, we report the bottom-up construction of a macromolecular transport system in which molecular pistons diffusively move through micrometer-long, hollow filaments. The pistons can cover micrometer distances in fractions of seconds. We build the system using multi-layer DNA origami and analyze the structures of the components using transmission electron microscopy. We study the motion of the pistons along the tubes using single-molecule fluorescence microscopy and perform Langevin simulations to reveal details of the free energy surface that directs the motions of the pistons. The tubular transport system achieves diffusivities and displacement ranges known from natural molecular motors and realizes mobility improvements over five orders of magnitude compared to previous artificial random walker designs. Electric fields can also be employed to actively pull the pistons along the filaments, thereby realizing a nanoscale electric rail system. Our system presents a platform for artificial motors that move autonomously driven by chemical fuels and for performing nanotribology studies, and it could form a basis for future molecular transportation networks.

DNA origami cryptography for secure communication.

Biomolecular cryptography exploiting specific biomolecular interactions for data encryption represents a unique approach for information security. However, constructing protocols based on biomolecular reactions to guarantee confidentiality, integrity and availability (CIA) of information remains a challenge. Here we develop DNA origami cryptography (DOC) that exploits folding of a M13 viral scaffold into nanometer-scale self-assembled braille-like patterns for secure communication, which can create a key with a size of over 700 bits. The intrinsic nanoscale addressability of DNA origami additionally allows for protein binding-based steganography, which further protects message confidentiality in DOC. The integrity of a transmitted message can be ensured by establishing specific linkages between several DNA origamis carrying parts of the message. The versatility of DOC is further demonstrated by transmitting various data formats including text, musical notes and images, supporting its great potential for meeting the rapidly increasing CIA demands of next-generation cryptography.

Solving mazes with single-molecule DNA navigators.

Molecular devices with information-processing capabilities hold great promise for developing intelligent nanorobotics. Here we demonstrate a DNA navigator system that can perform single-molecule parallel depth-first search on a ten-vertex rooted tree defined on a two-dimensional DNA origami platform. Pathfinding by the DNA navigators exploits a localized strand exchange cascade, which is initiated at a unique trigger site on the origami with subsequent automatic progression along paths defined by DNA hairpins containing a universal traversal sequence. Each single-molecule navigator autonomously explores one of the possible paths through the tree. A specific solution path connecting a given pair of start and end vertices can then be easily extracted from the set of all paths taken by the navigators collectively. The solution path laid out on origami is illustrated with single-molecule imaging. Our approach points towards the realization of molecular materials with embedded computational functions operating at the single-molecule level.

A DNA Nanorobot Uprises against Cancer.

DNA-based nanorobots have been shown to sense and respond to molecular triggers, such as intracellular pH and cell surface receptors. A recent report describes DNA nanorobots as potential cancer therapeutic agents that can be programmed to trigger coagulation inside blood vessels at the tumor site, starving tumor cells to death.

A self-assembled nanoscale robotic arm controlled by electric fields.

The use of dynamic, self-assembled DNA nanostructures in the context of nanorobotics requires fast and reliable actuation mechanisms. We therefore created a 55-nanometer-by-55-nanometer DNA-based molecular platform with an integrated robotic arm of length 25 nanometers, which can be extended to more than 400 nanometers and actuated with externally applied electrical fields. Precise, computer-controlled switching of the arm between arbitrary positions on the platform can be achieved within milliseconds, as demonstrated with single-pair Förster resonance energy transfer experiments and fluorescence microscopy. The arm can be used for electrically driven transport of molecules or nanoparticles over tens of nanometers, which is useful for the control of photonic and plasmonic processes. Application of piconewton forces by the robot arm is demonstrated in force-induced DNA duplex melting experiments.

Long-range movement of large mechanically interlocked DNA nanostructures.

Interlocked molecules such as catenanes and rotaxanes, connected only via mechanical bonds have the ability to perform large-scale sliding and rotational movements, making them attractive components for the construction of artificial molecular machines and motors. We here demonstrate the realization of large, rigid rotaxane structures composed of DNA origami subunits. The structures can be easily modified to carry a molecular cargo or nanoparticles. By using multiple axle modules, rotaxane constructs are realized with axle lengths of up to 355 nm and a fuel/anti-fuel mechanism is employed to switch the rotaxanes between a mobile and a fixed state. We also create extended pseudo-rotaxanes, in which origami rings can slide along supramolecular DNA filaments over several hundreds of nanometres. The rings can be actively moved and tracked using atomic force microscopy.

Diffusive transport of molecular cargo tethered to a DNA origami platform.

Fast and efficient transport of molecular cargoes along tracks or on supramolecular platforms is an important prerequisite for the development of future nanorobotic systems and assembly lines. Here, we study the diffusive transport of DNA cargo strands bound to a supramolecular DNA origami structure via an extended tether arm. For short distances (on the order of a few nanometers), transport from a start to a target site is found to be less efficient than for direct transfer without tether. For distances on the scale of the origami platform itself, however, cargo transfer mediated by a rigid tether arm occurs very fast and robust, whereas a more flexible, hinged tether is found to be considerably less efficient. Our results suggest diffusive motion on a molecular tether as a highly efficient mechanism for fast transfer of cargoes over long distances.

Robustness of localized DNA strand displacement cascades.

Colocalization can strongly alter the kinetics and efficiency of chemical processes. For instance, in DNA-templated synthesis unfavorable reactions are sped up by placing reactants into close proximity onto a DNA scaffold. In biochemistry, clustering of enzymes has been demonstrated to enhance the reaction flux through some enzymatic cascades. Here we investigate the effect of colocalization on the performance of DNA strand displacement (DSD) reactions, an important class of reactions utilized in dynamic DNA nanotechnology. We study colocalization by immobilizing a two-stage DSD reaction cascade comprised of a "sender" and a "receiver" gate onto a DNA origami platform. The addition of a DNA (or RNA) input strand displaces a signal strand from the sender gate, which can then transfer to the receiver gate. The performance of the cascade is found to vary strongly with the distance between the gates. A cascade with an intermediate gate distance of ≈20 nm exhibits faster kinetics than those with larger distances, whereas a cascade with smaller distance is corrupted by excessive intraorigami leak reactions. The 20 nm cascade is found to be considerably more robust with respect to a competing reaction, and implementation of multiple receiver gates further increases this robustness. Our results indicate that for the 20 nm distance a fraction of signal strands is transferred locally to a receiver gate on the same platform, probably involving direct physical contact between the gates. The performance of the cascade is consistent with a simple model that takes "local" and "global" transfer processes into account.