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.

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.

Riboswitch-inspired toehold riboregulators for gene regulation in Escherichia coli.

Regulatory RNA molecules have been widely investigated as components for synthetic gene circuits, complementing the use of protein-based transcription factors. Among the potential advantages of RNA-based gene regulators are their comparatively simple design, sequence-programmability, orthogonality, and their relatively low metabolic burden. In this work, we developed a set of riboswitch-inspired riboregulators in Escherichia coli that combine the concept of toehold-mediated strand displacement (TMSD) with the switching principles of naturally occurring transcriptional and translational riboswitches. Specifically, for translational activation and repression, we sequestered anti-anti-RBS or anti-RBS sequences, respectively, inside the loop of a stable hairpin domain, which is equipped with a single-stranded toehold region at its 5' end and is followed by regulated sequences on its 3' side. A trigger RNA binding to the toehold region can invade the hairpin, inducing a structural rearrangement that results in translational activation or deactivation. We also demonstrate that TMSD can be applied in the context of transcriptional regulation by switching RNA secondary structure involved in Rho-dependent termination. Our designs expand the repertoire of available synthetic riboregulators by a set of RNA switches with no sequence limitation, which should prove useful for the development of robust genetic sensors and circuits.

Rational design of hybrid DNA-RNA triplex structures as modulators of transcriptional activity in vitro.

Triplex nanostructures can be formed in vitro in the promoter region of DNA templates, and it is commonly accepted that these assemblies inhibit the transcription of the downstream genes. Herein, a proof of concept highlighting the possibility of the up- or downregulation of RNA transcription is presented. Hybrid DNA-RNA triplex nanostructures were rationally designed to produce bacterial transcription units with switchable promoters. The rate of RNA production was measured using the signal of a transcribed fluorescent RNA aptamer (i.e. Broccoli). Indeed, several designed bacterial promoters showed the ability of induced transcriptional inhibition, while other properly tailored sequences demonstrated switchable enhancement of transcriptional activity, representing an unprecedented feature to date. The use of RNA-regulated transcription units and fluorescent RNA aptamers as readouts will allow the realization of biocomputation circuits characterized by a strongly reduced set of components. Triplex forming RNA oligonucleotides are proposed as smart tools for transcriptional modulation and represent an alternative to current methods for producing logic gates using protein-based components.

Reversible Self-Assembly of Nucleic Acids in a Diffusiophoretic Trap.

The formation and dissociation of duplexes or higher order structures from nucleic acid strands is a fundamental process with widespread applications in biochemistry and nanotechnology. Here, we introduce a simple experimental system-a diffusiophoretic trap-for the non-equilibrium self-assembly of nucleic acid structures that uses an electrolyte gradient as the driving force. DNA strands can be concentrated up to hundredfold by a diffusiophoretic trapping force that is caused by the electric field generated by the electrolyte gradient. We present a simple equation for the field to guide selection of appropriate trapping electrolytes. Experiments with carboxylated silica particles demonstrate that the diffusiophoretic force is long-ranged, extending over hundreds of micrometers. As an application, we explore the reversible self-assembly of branched DNA nanostructures in the trap into a macroscopic gel. The structures assemble in the presence of an electrolyte gradient, and disassemble upon its removal, representing a prototypical adaptive response to a macroscopic non-equilibrium state.

Toehold-Mediated Strand Displacement in Random Sequence Pools.

Toehold-mediated strand displacement (TMSD) has been used extensively for molecular sensing and computing in DNA-based molecular circuits. As these circuits grow in complexity, sequence similarity between components can lead to cross-talk, causing leak, altered kinetics, or even circuit failure. For small non-biological circuits, such unwanted interactions can be designed against. In environments containing a huge number of sequences, taking all possible interactions into account becomes infeasible. Therefore, a general understanding of the impact of sequence backgrounds on TMSD reactions is of great interest. Here, we investigate the impact of random DNA sequences on TMSD circuits. We begin by studying individual interfering strands and use the obtained data to build machine learning models that estimate kinetics. We then investigate the influence of pools of random strands and find that the kinetics are determined by only a small subpopulation of strongly interacting strands. Consequently, their behavior can be mimicked by a small collection of such strands. The equilibration of the circuit with the background sequences strongly influences this behavior, leading to up to 1 order of magnitude difference in reaction speed. Finally, we compare two established and one novel technique that speed up TMSD reactions in random sequence pools: a three-letter alphabet, protection of toeholds by intramolecular secondary structure, or by an additional blocking strand. While all of these techniques were useful, only the latter can be used without sequence constraints. We expect that our insights will be useful for the construction of TMSD circuits that are robust to molecular noise.

Massively Parallel Double-Selection Workflow for the Evolution of Molecular Switches Based on Surface-Display in Escherichia coli.

Microbial surface display of proteins is a versatile method for a wide range of biotechnological applications. Herein, the use of a surface display system in E. coli for the evolution of a riboswitch from an RNA aptamer is presented. To this end, a streptavidin-binding peptide (SBP) is displayed at the bacterial surface, which can be used for massively parallel selection using a magnetic separation system. Coupling gene expression from a riboswitch library to the display of SBP hence allows selection of library members that express strongly in the presence of a ligand. As excessive SBP expression leads to bacterial growth inhibition, it can be used to negatively select against leaky riboswitches expressing in the absence of ligand. Based on this principle, we devise a double selection workflow that enables quick selection of functional riboswitches with a comparatively low screening workload. The efficiency of our protocol by re-discovering a previously isolated theophylline riboswitch from a library was demonstrated, as well as a new riboswitch that is similar in performance, but slightly more responsive at low theophylline concentrations. Our workflow is massively parallel and can be applied to the screening or pre-screening of large molecular libraries.

Nucleic acid strand displacement - from DNA nanotechnology to translational regulation.

Nucleic acid strand displacement reactions involve the competition of two or more DNA or RNA strands of similar sequence for binding to a complementary strand, and facilitate the isothermal replacement of an incumbent strand by an invader. The process can be biased by augmenting the duplex comprising the incumbent with a single-stranded extension, which can act as a toehold for a complementary invader. The toehold gives the invader a thermodynamic advantage over the incumbent, and can be programmed as a unique label to activate a specific strand displacement process. Toehold-mediated strand displacement processes have been extensively utilized for the operation of DNA-based molecular machines and devices as well as for the design of DNA-based chemical reaction networks. More recently, principles developed initially in the context of DNA nanotechnology have been applied for the de novo design of gene regulatory switches that can operate inside living cells. The article specifically focuses on the design of RNA-based translational regulators termed toehold switches. Toehold switches utilize toehold-mediated strand invasion to either activate or repress translation of an mRNA in response to the binding of a trigger RNA molecule. The basic operation principles of toehold switches will be discussed as well as their applications in sensing and biocomputing. Finally, strategies for their optimization will be described as well as challenges for their operation in vivo.

Switchable Fluorescent Light-Up Aptamers Based on Riboswitch Architectures.

Fluorescent light-up RNA aptamers (FLAPs) such as Spinach or Mango can bind small fluorogens and activate their fluorescence. Here, we adopt a switching mechanism otherwise found in riboswitches and use it to engineer switchable FLAPs that can be activated or repressed by trigger oligonucleotides or small metabolites. The fluorophore binding pocket of the FLAPs comprises guanine (G) quadruplexes, whose critical nucleotides can be sequestered by corresponding anti-FLAP sequences, leading to an inactive conformation and thus preventing association with the fluorophore. We modified the FLAPs with designed toehold hairpins that carry either an anti-FLAP or an anti-anti-FLAP sequence within the loop region. The addition of an input RNA molecule triggers a toehold-mediated strand invasion process that refolds the FLAP into an active or inactive configuration. Several of our designs display close-to-zero leak signals and correspondingly high ON/OFF fluorescence ratios. We also modified purine aptamers to sequester a partial anti-FLAP or an anti-anti-FLAP sequence to control the formation of the fluorogen-binding conformation, resulting in FLAPs whose fluorescence is activated or deactivated in the presence of guanine or adenine. We demonstrate that switching modules can be easily combined to generate FLAPs whose fluorescence depends on several inputs with different types of input logic.

Tunable 2D diffusion of DNA nanostructures on lipid membranes.

DNA nanotechnology facilitates the synthesis of biomimetic models for studying biological systems. This work uses lipid bilayers as platforms for two-dimensional single-particle tracking of the dynamics of DNA nanostructures. Three different DNA origami structures adhere to the membrane through hybridization with cholesterol-modified strands. Their two-dimensional diffusion coefficient is modulated by changing the concentration of monovalent and divalent salts and the number of anchors. In addition, the diffusion coefficient is tuned by targeting cholesterol-modified anchor strands with strand-displacement reactions. We demonstrate a responsive system with changing diffusivity by selectively displacing membrane-bound anchor strands. We also show the programmed release of origami structures from the lipid membranes.

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.

Emergence of Colloidal Patterns in ac Electric Fields.

Suspended microparticles subjected to ac electrical fields collectively organize into band patterns perpendicular to the field direction. The bands further develop into zigzag shaped patterns, in which the particles are observed to circulate. We demonstrate that this phenomenon can be observed quite generically by generating such patterns with a wide range of particles: silica spheres, fatty acid, oil, and coacervate droplets, bacteria, and ground coffee. We show that the phenomenon can be well understood in terms of second order electrokinetic flow, which correctly predicts the hydrodynamic interactions required for the pattern formation process. Brownian particle simulations based on these interactions accurately recapitulate all of the observed pattern formation and symmetry-breaking events, starting from a homogeneous particle suspension. The emergence of the formed patterns can be predicted quantitatively within a parameter-free theory.

Evaluation of an E. coli Cell Extract Prepared by Lysozyme-Assisted Sonication via Gene Expression, Phage Assembly and Proteomics.

Over the past decades, starting from crude cell extracts, a variety of successful preparation protocols and optimized reaction conditions have been established for the production of cell-free gene expression systems. One of the crucial steps during the preparation of cell extract-based expression systems is the cell lysis procedure itself, which largely determines the quality of the active components of the extract. Here we evaluate the utility of an E. coli cell extract, which was prepared using a combination of lysozyme incubation and a gentle sonication step. As quality measure, we demonstrate the cell-free expression of YFP at concentrations up to 0.6 mg/mL. In addition, we produced and assembled T7 bacteriophages up to a titer of 108  PFU/mL. State-of-the-art quantitative proteomics was used to compare the produced extracts with each other and with a commercial extract. The differences in protein composition were surprisingly small between lysozyme-assisted sonication (LAS) extracts, but we observed an increase in the release of DNA-binding proteins for increasing numbers of sonication cycles. Proteins taking part in carbohydrate metabolism, glycolysis, amino acid and nucleotide related pathways were found to be more abundant in the LAS extract, while proteins related to RNA modification and processing, DNA modification and replication, transcription regulation, initiation, termination and the TCA cycle were found enriched in the commercial extract.

De novo-designed translation-repressing riboregulators for multi-input cellular logic.

Efforts to construct synthetic biological circuits with more complex functions have often been hindered by the idiosyncratic behavior, limited dynamic range and crosstalk of commonly utilized parts. Here, we employ de novo RNA design to develop two high-performance translational repressors with sensing and logic capabilities. These synthetic riboregulators, termed toehold repressors and three-way junction (3WJ) repressors, detect transcripts with nearly arbitrary sequences, repress gene expression by up to 300-fold and yield orthogonal sets of up to 15 devices. Automated forward engineering is used to improve toehold repressor dynamic range and SHAPE-Seq is applied to confirm the designed switching mechanism of 3WJ repressors in living cells. We integrate the modular repressors into biological circuits that execute universal NAND and NOR logic and evaluate the four-input expression NOT ((A1 AND A2) OR (B1 AND B2)) in Escherichia coli. These capabilities make toehold and 3WJ repressors valuable new tools for biotechnological applications.

Principles and Applications of Nucleic Acid Strand Displacement Reactions.

Dynamic DNA nanotechnology, a subfield of DNA nanotechnology, is concerned with the study and application of nucleic acid strand-displacement reactions. Strand-displacement reactions generally proceed by three-way or four-way branch migration and initially were investigated for their relevance to genetic recombination. Through the use of toeholds, which are single-stranded segments of DNA to which an invader strand can bind to initiate branch migration, the rate with which strand displacement reactions proceed can be varied by more than 6 orders of magnitude. In addition, the use of toeholds enables the construction of enzyme-free DNA reaction networks exhibiting complex dynamical behavior. A demonstration of this was provided in the year 2000, in which strand displacement reactions were employed to drive a DNA-based nanomachine (Yurke, B.; et al. Nature 2000, 406, 605-608). Since then, toehold-mediated strand displacement reactions have been used with ever increasing sophistication and the field of dynamic DNA nanotechnology has grown exponentially. Besides molecular machines, the field has produced enzyme-free catalytic systems, all DNA chemical oscillators and the most complex molecular computers yet devised. Enzyme-free catalytic systems can function as chemical amplifiers and as such have received considerable attention for sensing and detection applications in chemistry and medical diagnostics. Strand-displacement reactions have been combined with other enzymatically driven processes and have also been employed within living cells (Groves, B.; et al. Nat. Nanotechnol. 2015, 11, 287-294). Strand-displacement principles have also been applied in synthetic biology to enable artificial gene regulation and computation in bacteria. Given the enormous progress of dynamic DNA nanotechnology over the past years, the field now seems poised for practical application.

Controlling Gene Expression in Mammalian Cells Using Multiplexed Conditional Guide RNAs for Cas12a*.

Spatiotemporal control of the activity of CRISPR-associated (Cas) proteins is of considerable interest for basic research and therapeutics. Here, we show that conditional guide RNAs (gRNAs) for Cas12a can be transcribed in mammalian cells by RNA polymerase II, followed by activation via input-dependent processing of the 3' tail of the gRNA transcript. We demonstrate processing using an RNA strand displacement mechanism, as well as microRNA-dependent processing, and cleavage by a guanine-responsive ribozyme. We further demonstrate that Cas12a along with several independently switchable gRNAs can be compactly integrated on a single transcript using stabilizing RNA triplexes, providing a route towards Cas12a-based gene regulation constructs with multi-input switching capabilities. The principle is shown to work in HEK and mouse fibroblast cells using luminescence, fluorescence, and is also demonstrated for the conditional upregulation of an endogenous gene.

Programming Diffusion and Localization of DNA Signals in 3D-Printed DNA-Functionalized Hydrogels.

Additive manufacturing enables the generation of 3D structures with predefined shapes from a wide range of printable materials. However, most of the materials employed so far are static and do not provide any intrinsic programmability or pattern-forming capability. Here, a low-cost 3D bioprinting approach is developed, which is based on a commercially available extrusion printer that utilizes a DNA-functionalized bioink, which allows to combine concepts developed in dynamic DNA nanotechnology with additive patterning techniques. Hybridization between diffusing DNA signal strands and immobilized anchor strands can be used to tune diffusion properties of the signals, or to localize DNA strands within the gel in a sequence-programmable manner. Furthermore, strand displacement mechanisms can be used to direct simple pattern formation processes and to control the availability of DNA sequences at specific locations. To support printing of DNA-functionalized gel voxels at arbitrary positions, an open source python script that generates machine-readable code (GCODE) from simple vector graphics input is developed.

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.

Growth of Giant Peptide Vesicles Driven by Compartmentalized Transcription-Translation Activity.

Compartmentalization and spatial organization of biochemical reactions are essential for the establishment of complex metabolic pathways inside synthetic cells. Phospholipid and fatty acid membranes are the most natural candidates for this purpose, but also polymers have shown great potential as enclosures of artificial cell mimics. Herein, we report on the formation of giant vesicles in a size range of 1 µm-100 µm using amphiphilic elastin-like polypeptides. The peptide vesicles can accommodate cell-free gene expression reactions, which is demonstrated by the transcription of a fluorescent RNA aptamer and the production of a fluorescent protein. Importantly, gene expression inside the vesicles leads to a strong growth of their size-up to an order of magnitude in volume in several cases-which is driven by changes in osmotic pressure, resulting in fusion events and uptake of membrane peptides from the environment.

Controlling Chirality across Length Scales using DNA.

Nano-objects with chiral properties attract growing interest due to their relevance for a wide variety of technological applications. For example, chiral nano-objects may be used in characterization platforms that involve chiral molecular recognition of proteins or in the fabrication of nanomechanical devices such as screw-gears or nanoswimmers. Spatial ordering of emitters of circularly polarized light might greatly benefit from the utilization of chiral shapes. Tools developed in DNA nanotechnology now allow precise tailoring of the chiral properties of molecules and materials at various length scales. Among others, they have already been applied to control the handedness of helical shapes (configurational chirality) or the chiral positioning of different-sized nanoparticles at the vertices of tetrahedra (compositional chirality). This work covers some of the key advances and recent developments in the field of chiral DNA nanoarchitectures and discusses their future perspectives and potential applications.