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Constructing and powering nanoscale DNA origami motors

Periodic Reporting for period 3 - DNA ORIGAMI MOTORS (Constructing and powering nanoscale DNA origami motors)

Reporting period: 2020-05-01 to 2021-10-31

Creating autonomously running motor units capable of driving directed transport against external loads is a key unmet challenge in nanoscale science and technology. Researchers have identified fundamental physical requirements necessary to achieve directional motion (1-3), including that motors 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 (4-10), 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 the function of natural macromolecular machines. One difficulty hindering unravelling these principles is that natural macromolecular structures often carry evolutionary ballast that may obscure the principles driving their functioning. Artificial nanoscale motors 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. Recent advances in creating complex three-dimensional (3D) structures using the methods of structural DNA nanotechnology (11-16) have created 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 are:
(1) Extend our preliminary work with a 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.
(2) Experimentally test various out-of-equilibrium physical methods to achieve directed motion in ratchet- like mechanisms, including thermal or flashing ratchet methods (2), asymmetric dissipative fluxes, and coupling mechanisms to chemical reactions.
(3) Produce translational motors that can take thousands of consecutive steps on linear tracks, can move with speeds up to ~ 1 μm / s, are capable of operating against piconewton-scale backward forces; and produce rotary motors that rotate with ~1000 revolutions per second, creating torques on the 10 pN*nm scale. To date, only natural protein motors can operate at this speed and efficiency.
(4) 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 drive chemical reactions out-of-equilibrium.
In this funding period we successfully accomplished and published the construction of synthetic molecular mechanisms with rotational and translational degrees of freedom (Stömmer et al, A synthetic tubular molecular transport system, Nature Communications 2021; Bertosin et al, A nanoscale reciprocating rotary mechanism with coordinated mobility control, Nature Communications 2021). These systems, although passively driven by random Brownian motions and not capable of any directed motions, were important stepping stones for us to establish synthesis methods and analytical methods for studying these systems. In addition, we constructed 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. Satisfyingly, we did succeed! We 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 thus capable of doing work on the environment. We are currently in the process of writing up manuscripts to report these successes to the scientific community, and we plan to expand scientific inquiry into these novel types of electrical motors. On the other hand, we discarded thermal ratcheting as a viable mode of action. In addition to this core work, we also pursued several other avenues of inquiry, such as logic-gated switching of DNA nanodevices (published in Dec 2021 in JACS, Engelen et al); and in-depth structural analysis of DNA nanostructures by cryo-EM (Kube et al, 2020, Nature Communications; Bertosin et al, 2021 ACS Nano). The experimental laboratory activities were naturally affected somewhat through lockdowns and social distancing measures in response to the COVID-19 pandemics.
The proposed project is likely to provide key advances that will impact 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 has the potential to provide such units. Second, the construction of artificial nanoscale motors provides an interesting arena to experimentally study transport phenomena occurring far from equilibrium on the nanoscale. Third, both the process of building such functional motor units and the resultant units themselves could help uncover previously unrecognized design principles underlying the function of natural macromolecular machines. One reason these these principles have been difficult to untangle is that natural macromolecular structures often carry evolutionary ballast that may obscure them. Artificial nanoscale motors could help explore in particular how mechanical motion may be coupled to chemical reactions. Fourth, 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