Molecular shuttles have been built from motor proteins capable of moving cargo along engineered paths. We illustrate alternative methods of controlling the direction of motion of microtubules on engineered kinesin tracks, how to load cargo covalently to microtubules, and how to exploit UV-induced release of caged ATP combined with enzymatic ATP degradation by hexokinase to turn the shuttles on and off sequentially. These are the first steps in the development of a tool kit to utilize molecular motors for the construction of nanoscale assembly lines.One of the central challenges in nanotechnology is how to assemble nanoscale building blocks including molecular wires and nanoscale switches into addressable devices, where the building blocks can be either synthesized, self-assembled in solution, or fabricated at the nanoscale. Ideally, a nanoscale conveyor belt would allow us to transport a selected nanoscale item over a defined distance and then facilitate the docking under defined angles with a second item of choice. One labor-intensive approach is to use tips of scanning probe microscopes to pick up, move, and release atoms, single molecules, and nanoscale objects in a controlled manner, one at a time. 1 Nanoscale objects created in this way are therefore pieces of art rather than devices of economical value. Methods need to be developed that allow the parallel assembly of nanoscale building blocks in large numbers. Here we combine methods of micro-and nanofabrication, with molecular assembly, and molecular motors powered chemically by adenosine triphosphate (ATP) to construct molecular shuttles that move under user control. This represents the first step in learning how to construct and operate nanoscale conveyor belts in large numbers.Key features of conveyor belts are that they (a) are driven by a force-generating motor, (b) transport cargo unidirectionally between well-defined positions, (c) accommodate loading and docking of cargo, and (d) can be externally switched on and off. These features are independent of the length scale at which conveyor belts are constructed and operated. If operation at the nanoscale is desired, molecular motors are required to generate force to propel cargo. Furthermore, schemes have to be developed to guide molecular movement along precisely controlled tracks, to load and unload cargo, and to control the speed of motion noninvasively. We will review and describe novel approaches that can serve as universal elements of a tool kit to construct and operate molecular shuttles. Molecular Motors. Nature has evolved unique molecules, termed motor proteins, that transport cargo over long distances or rotate loads in an energy-dependent manner. The characteristics of these motor proteins are far superior to all demonstrated miniaturized synthetic engines. 2 Motor proteins generate more force, have better fuel efficiency, and are smaller in size than any man-made device.

Cells regulate active transport of intracellular cargo using motor proteins. Recent nanobiotechnology efforts aim to adapt motor proteins to power the movement and assembly of synthetic materials. A motor-protein-based nanoscale transport system (molecular shuttle) requires that the motion of the shuttles be guided along tracks. This study investigates the principles by which microtubules, serving as shuttle units, are guided along micrometer-scale kinesin-coated chemical and topographical tracks, where the efficiency of guidance is determined by events at the track boundary. Thus, we measure the probability of guiding as microtubules reach the track boundary of (1) a chemical edge between kinesin-coated and kinesin-free surfaces, (2) a topography-only wall coated completely with kinesin, and (3) a kinesin-free wall next to a kinesin-coated bottom surface (topography and chemistry combined). We present a guiding mechanism for each surface type that takes into account the physical properties of microtubule filaments and the surface properties (geometry, chemistry), and elucidate the contributions of surface topography and chemistry. Our experimental and theoretical results show that track edges that combine both topography and chemistry guide microtubules most frequently (approximately 90% of all events). By applying the principles of microtubule guidance by microfabricated surfaces, one may design and build motor-protein-powered devices optimized for transport.

Mastering supramolecular self-assembly to a similar degree as nature has achieved on a subcellular scale is critical for the efficient fabrication of complex nanoscopic and mesoscopic structures. We demonstrate that active, molecular-scale transport powered by biomolecular motors can be utilized to drive the self-assembly of mesoscopic structures that would not form in the absence of active transport. In the presented example, functionalized microtubules transported by surface-immobilized kinesin motors cross-link via biotin/streptavidin bonds and form extended linear and circular mesoscopic structures, which move in the presence of ATP. The self-assembled structures are oriented, exhibit large internal strains, and are metastable while the biomolecular motors are active. The integration of molecular motors into the self-assembly process overcomes the trade-off between stability and complexity in thermally activated molecular self-assembly.