Periodic Reporting for period 4 - DNA ORIGAMI MOTORS (Constructing and powering nanoscale DNA origami motors) Reporting period: 2021-11-01 to 2022-04-30 Summary of the context and overall objectives of the project Creating autonomously running motor units capable of driving directed transport against external loads is a key unmet challenge in nanoscale science and technology. The fundamental physical requirements necessary to achieve directional motion include that motor elements must diffuse within an asymmetric periodic energy landscape under conditions far from thermal equilibrium as created by stochastic or deterministic energy input. However, the practical implementation of these principles on the nanoscale requires extraordinary control over the structure of matter. While much progress has been made in the bottom-up synthesis of molecular and macromolecular mechanisms, including the creation of prototypical objects having machine- or motor-like properties, the construction of synthetic motors that can work against external forces and move at speeds and with efficiencies rivalling those of natural motor proteins still remains elusive. Construction of artificial nanoscale motors could help explore and test experimentally physical theories of transport phenomena occurring far from equilibrium. Both the process of building such functional motor units and the resultant units themselves could help uncover previously unrecognized design principles underlying for driving macromolecular machines. Construction artificial nanoscale motors according to principles of rational design could help elucidate in particular how mechanical motion may be coupled to chemical reactions. Finally, robustly functioning artificial motor units could be of great practical use in nanotechnology, for example to drive chemical synthesis, to actively propel nanoscale drug delivery vehicles, to pump and separate molecules across barriers or to package molecules into cargo components. Advances in our capabilities to fabricate complex three-dimensional (3D) structures using the methods of structural DNA nanotechnology have created new opportunities to take the next step and pursue construction of nanoscale rotary and translational devices, as well as to test various candidate methods for powering directed motion in these devices. Our specific objectives in this project included building prototypical rotary mechanism having fast rotary mobility to create structurally well-defined diffusive mechanisms based on tight-fitting nanoscale 3D components that have defined translational or rotational degrees of freedom, experimentally testing various out-of-equilibrium physical methods to achieve directed motion in ratchet-like mechanisms, including thermal or flashing ratchets, and explore how directional motor movement on the nanoscale as induced by macroscopically controlled energy input (e.g physical flashing ratchet methods or by directional fluxes) may be harnessed to perform mechanical work.We indeed successfully accomplished our ultimate goal and succeeded developing three distinct DNA-origami based molecular motor systems. Two of these three devices have been presented in papers published in major scientific journals (Pumm et al, A DNA origami rotary ratchet motor, Nature 2022 and Shi et al, Nature Physics 2022). In the quest to build nanoscale motors, this project was therefore able to provide breakthrough advances. We successfully produced prototypes of nanomotors that run processively, autonomously, and with multiple orders of magnitudes faster speeds than any previously created artificial molecular motor. Our motors even can generate measurable torque, which we demonstrate for example by using the motors to wind up a molecular spring, and then let the motors run driven by the energy stored in a spring. For the first time, an artificial motor system now exists with mechanical capabilities that approach those of celebrated biological motors such as the F1 ATPase. Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far During the first and second reporting period (RP1, RP2) multiple generations of rotary mechanisms with ratchet implementations and also translational mechanisms were being designed. We also discovered an attractive possibility to covalently stabilize DNA origami nanostructures. This is highly advantageous for this project, as it allows us to build and test machine-like objects for a much wider range of conditions. The additional covalent bonds may be used in a sequence-programmable fashion to link free strand termini, to bridge strand breaks at crossover sites, and to create additional interhelical connections. We built several new DNA origami prototypes and characterized them using single particle cryo-EM. We started to carry out single-molecule fluorescence experiments to study the motion of these mechanisms. A prototype of a translational mechanism with long-range travel along a one-dimensional track was successfully prepared. We also developed design tools to construct scaffold sequences de novo based on strand diagrams, and we developed scalable production methods for creating design-specific scaffold strands with fully user-defined sequences.In the third funding period (RP3) we successfully accomplished and published the construction and study of synthetic molecular mechanisms with rotational and translational degrees of freedom. These systems, although still being passive mechanisms displaying only undirected random Brownian motions, were important stepping stones for us to establish synthesis methods and analytical methods for studying the dynamics and mechanical interplay of the components in these systems. Yet, we also succeeded constructing several motor prototypes with rotary degrees of freedom that we tested for motor activity in response of different stimuli, notably ion currents through solid state nanopores, and by alternating AC fields, which we could successfully drive directionally. We thus now have two prototypes of actual molecular motors, a nanoscale turbine, and a nanoscale electrical motor that both show directional motion and sustained motion against viscous drag. The motors are capable of doing work on the environment. Much of RP3 was then consumed by the process of completing the in depth study of these new motors prototypes. The last funding period (RP4) covered only a short time frame (6 months), but we completed demonstration of three novel and distinct DNA-origami based molecular motor systems. These three systems are capable of directed motions and doing work on the environment, as evidenced by their sustained directional motion against viscous friction. We also directly show the ability to do work by winding a molecular spindle with our electrically driven DNA origami nanomotor. With angular speeds up to 250 revolutions per minute and torques up to 10 pN nm, the motors achieve rotational speeds and torques that are approaching those known from powerful natural molecular machines, such as the ATP synthase. The motors move directionally owing to intrinsic mechanistic properties, powered by a simple external energy modulation that does not need any feedback or information supplied by the user to direct the motors. Our motors also afford control options that one is familiar with from macroscale motors: the user can turn them on and off at will, they respond quickly, and the speed and direction of rotation can be regulated. The motors can be produced and operated by anyone having access to standard wet-lab equipment. It suffices to transmit the sequence information to enable other users to replicate and build their own motors using DNA molecules obtained, for example, from commercial sources. Owing to the modularity of the DNA origami components, we expect that the motors can also be modified, adapted and integrated into other contexts. A natural next frontier would be to explore causing a barrier modulation through a chemical reaction and to exploit the directed motor motion to drive uphill chemical synthesis using a more elaborate synthetic mechanism featuring coordinated reciprocal motion—much like F1F0-ATP synthase mechanically synthesizes ATP driven by rotary motion. In addition to this core work, we also pursued several other avenues of inquiry, such as logic-gated switching of DNA nanodevices; and in-depth structural analysis of DNA nanostructures by cryo-EM. The experimental laboratory activities were naturally affected somewhat through lockdowns and social distancing measures in response to the COVID-19 pandemics. Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far) The project provided key advances impacting a number of different fields. First, DNA-based nanotechnology has advanced rapidly but lacks efficient motor units that can transduce macroscopically applied energy input into directed nanoscale motions that can power other processes. Our project now provided three new, distinct molecular motor units. These motors provide an excellent basis for further study, for example to implement chemically-driven motors, or to exploit the mechanical rotation to drive chemical reactions uphill, much like ATP synthase does it. Second, the construction of artificial nanoscale motors provides an interesting arena to experimentally study transport phenomena occurring far from equilibrium on the nanoscale. For example, in one of our key studies we prove that the motors operate out of equilibrium by verifying that its entropy production satisfies the fluctuation theorem, which generalizes the second law of thermodynamics to far from equilibrium systems at the nanoscale. We believe this is the first experimental demonstration of this fundamental property of non-equilibrium systems in an artificial nanomotor. Third, to realize our motors we developed new means of assembly and also new means of validating the structures of these assemblies using single-particle cryo electron microscopy, which may be beneficial for building other, yet more complex devices in the future. final-artwork.jpg
