Nanoscale Transport Enables Active Self-Assembly of Millimeter- Scale Wires Ofer Idan, Amy Lam, Jovan Kamcev, John Gonzales, Ashutosh Agarwal, and Henry Hess* Department of Biomedical Engineering, Columbia University, New York, New York, United States * S Supporting Information ABSTRACT: Active self-assembly processes exploit an energy source to accelerate the movement of building blocks and intermediate structures and modify their interactions. A model system is the assembly of biotinylated microtubules partially coated with streptavidin into linear bundles as they glide on a surface coated with kinesin motor proteins. By tuning the assembly conditions, microtubule bundles with near millimeter length are created, demonstrating that active self-assembly is beneficial if components are too large for diffusive self-assembly but too small for robotic assembly. KEYWORDS: Self-assembly, active transport, biomolecular motor, nanowire, kinesin, microtubule T he integration of molecular and nanoscale components into functional macroscopic systems is one of the central challenges in nanotechnology. On the bottom-up route, the challenge arises from the unfavorable scaling of diffusion-driven self-assembly with increasing component size. 1,2 On the top- down route, the sequential nature of robotic assembly is ill- suited to the high throughput required by the large number of microscopic components. 3 Natural growth processes, e.g., embryogenesis, assemble complex macroscopic structures by using motile cells which position themselves using active, energy-consuming movement. 4 Endowing molecular and nano- scale building blocks with the ability to autonomously move, e.g., by loading them onto molecular shuttles, may similarly overcome the limitations of diffusion-driven self-assembly. Here it is shown experimentally that active transport by biomolecular motors can assemble structures of nearly 1 mm in length from building blocks only about 10 μm in size. A basic theoretical model of the assembly process illustrates the advantages and limitations of active transport processes relative to diffusion-driven self-assembly and robotic assembly. The experimental model system is the assembly of extended bundles of biotinylated microtubules cross-linked by streptavi- din via active transport on a kinesin-coated surface (Figure 1). Microtubules are cytoskeletal filaments with a diameter of 25 nm assembled from thousands of tubulin subunits. 5 When polymerized in vitro, their lengths are described by a Schulz distribution with an average length on the order of 5 μm. 6 Kinesin motor proteins adhered to a surface can transport microtubules with a velocity of hundreds of nanometers per second 7,8 on a trajectory which can be described by a worm-like chain model with a persistence length of 0.1 mm. 9 Biotinylated microtubules can be cross-linked with streptavidin. When cross- linking occurs during microtubule gliding on kinesin-coated surfaces, extended linear bundles and spools of microtubules are formed. 10,11 This process is a striking example of an active self-assembly process 12 and has been investigated in several recent studies. 13−20 Here we show that by optimizing the microtubule transport velocity, which affects the time available for biotin−streptavidin binding 21 and reduces spool forma- tion, 18 microtubule bundles with near millimeter lengths can be formed. This represents a 10-fold increase in the reported size of the assembled microtubule structures. 22 In our experiments, biotinylated microtubules were adsorbed to kinesin-coated surfaces and exposed to a 10 nM streptavidin solution for 5 min. This leads to partial coverage of the biotin linkers on the microtubules, enabling cross-linking when microtubules collide. Varying the microtubule gliding velocity by adjusting kinesin motor activity via the ATP concentration in the solution affected the length of the longest observed microtubule bundles. The ATP concentration was maintained at a constant level for hours by employing an ATP regenerating system. 23 Otherwise, the assembly process is arrested at low ATP concentrations by a lack of ATP within an hour. ATP concentrations of 20 μM (corresponding to gliding speeds of 0.1 μm/s) proved optimal (Supporting Information, Figure S1). Collisions of the gliding microtubules lead to the formation of bundles, which collide with each other and form even larger linear bundles. Bundles are converted into spools when simultaneous collisions of multiple bundles create circular topologies or when the tip of the bundle encounters a defective motor and its movement is arrested leading to a spiraling movement and the formation of a spool. 17 Therefore, bundles are transient structures (unless gliding is stopped by removal of ATP or chemical fixation) and require a suitable combination of initial microtubule densities, streptavidin concentrations, Received: October 3, 2011 Revised: November 21, 2011 Published: November 24, 2011 Letter pubs.acs.org/NanoLett © 2011 American Chemical Society 240 dx.doi.org/10.1021/nl203450h | Nano Lett. 2012, 12, 240−245 Downloaded by STANFORD UNIV at 17:30:40:535 on July 01, 2019 from https://pubs.acs.org/doi/10.1021/nl203450h.