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Constructing and powering nanoscale DNA origami motors

Periodic Reporting for period 2 - DNA ORIGAMI MOTORS (Constructing and powering nanoscale DNA origami motors)

Reporting period: 2018-11-01 to 2020-04-30

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.
We had proposed design and self assembly of improved rotary motor units including asymmetric ratchet like features. Several new prototypes have been successfully built, and characterized structurally using single particle cryo EM. Furthermore, we have begun to carry out single-molecule fluorescence experiments to study the motion of these mechanisms. Also, we have proposed translational mechanisms, and we have also successfully prepared a working prototype with long-range travel along a one-dimensional track. None of these prototypes has been presented in the public so far. Once we have completed single particle mobility measurements in and out of thermal equilibrium, we will aim for publishing the results. Furthermore, efforts are underway to develop new catalytic DNAzymes to drive conformational changes in the mechanisms while substrate molecules are being consumed

In accordance with the proposed timeline, we have added a heating system to our single-molecule microscope to be able to periodically control the temperature of the particles. In parallel, we have a established another setup for single-molecule interference scattering contrast measurements, which allows studying single particle dynamics with much higher frame rates and without the observation time scale limitations imposed by bleaching of fluorescent dyes as in conventional fluorescence microscopy. Some preliminary results with this technique were already published (Häussermann et al, Angewandte Chemie 2019).

We have also realized that successful motor production required much improvided particle stabilization and would also benefit from the possibility for reversible covalent attachments. Therefore we have developed a sequence-programmable method for crosslinking DNA nanostructures using UV light, and we have begun to synthesize a variety of modified DNA bases containing light actuatable moeties (azobenzene for motion, CNVK for reversible bonding). Part of these efforts have already been published (Gerling et al, Science Advances 2018; Gerling et al, Angewandte Chemie 2019).

We have invested substantial effort in better understanding folding requirements for DNA origami. To this end, we measured the folding kinetics of every staple strand and its two terminal segments during constant-temperature assembly of a multilayer DNA origami object. Our data illuminate the processes occurring during folding of the DNA origami in fine detail, starting with the first nucleating double-helical domains and ending with the fully folded DNA origami object. We found a complex sequence of folding events that cannot be explained with sim- plistic local design analysis. Our real-time data, although derived from one specific DNA origami object, through its sheer massive detail, could provide the crucial input needed to construct and test a quantitatively predictive, general model of DNA origami assembly. These results were also published (Schneider et al, Science Advances 2019).

DNA origami nano-objects are usually designed around generic single-stranded “scaffolds”. Many properties of the target object are determined by details of those generic scaffold sequences. Here, we enable designers to fully specify the target structure not only in terms ofdesired 3D shape but also in terms of the sequences used. To this end, we built 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. We used 17 custom scaffolds having different lengths and sequence properties to study the influence of sequence redundancy and sequence composition on multilayer DNA origami assembly and to realize efficient one-pot assembly of multiscaffold DNA origami objects. Furthermore, as examples for functionalized scaffolds, we created a scaffold that enables direct, covalent cross-linking of DNA origami via UV irradiation, and we built DNAzyme-containing scaffolds that allow postfolding DNA origami domain separation. These results were published (Engelhardt et al, ACS Nano 2019)
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