Biotemplated Nanopatterning of Planar Surfaces with Molecular Motors Cordula Reuther, ² Lukasz Hajdo, ‡ Robert Tucker, § Andrzej A. Kasprzak, ‡ and Stefan Diez* ,² Max-Planck-Institute of Molecular Cell Biology and Genetics, Pfotenhauerstrasse 108, 01307 Dresden, Germany, The Nencki Institute of Experimental Biology, Pasteur 3, 02-093 Warsaw, Poland, and UniVersity of Florida, GainsVille, Department of Materials Science and Engineering, 160 Rhines Hall, GainesVille, Florida 32611-6400 Received April 25, 2006; Revised Manuscript Received July 27, 2006 ABSTRACT We report on the generation of nanometer-wide, non-topographical patterns of proteins on planar surfaces. In particular, we used the regular lattice of reconstituted microtubules as template structures to specifically bind and transfer kinesin-1 and nonclaret disjunctional motor proteins. The generated tracks, which comprise dense and structurally oriented arrays of functional motor proteins, proved to be highly efficient for the guiding of microtubule transporters. Biomolecular motors are currently explored for an increasing number of applications in hybrid bionanodevices. 1 Along these lines, gliding motility assays, where reconstituted microtubule filaments are propelled over a substrate by surface-attached motor proteins, have been used to transport micrometer- and nanometer-sized objects, such as small beads, 2 quantum dots 3 or DNA molecules. 4 However, one prerequisite for controllable nanotransport is the reliable guiding of filament movement along predefined paths, a challenging task that has recently been achieved only via costly and labor-intensive topographical surface modifica- tions. 2,5-10 Here, we report on the generation of nanometer- wide, non-topographical tracks of motor proteins. In par- ticular, we used the regular lattice of reconstituted micro- tubules as template structures to specifically bind and transfer kinesin-1 and nonclaret disjunctional (Ncd) motor proteins on planar surfaces. Through this approach, which has been inspired by biological transport systems found within cells, dense and structurally oriented arrays of functional motor proteins were created. The motor tracks proved to be highly efficient for the guiding of microtubule transporters. Microtubules are cytoskeletal filaments 25 nm in diameter and several micrometers long. Their lattice displays an 8 nm periodicity originating from the size of the dimeric tubulin proteins that make up the protofilaments. We investigated two different methods of biotemplated nanopatterning of planar surfaces with motor proteins: “biotemplated stamp- ing” (Figure 1a) and “biotemplated binding” (Figure 1b). In the stamping approach, kinesin-1 molecules were bound in solution with their motor domains to “template” microtubules in the absence of ATP. The generated complexes were then adsorbed onto the surface (step I), and ATP was added in order to propel the template microtubules off the surface- bound motor proteins. This way, tracks of oriented motor molecules, with their motor domains pointing away from the surface, were generated (step II). In the binding approach, the template microtubules were first immobilized on the surface (step I). Kinesin-1 or Ncd motor proteins were then specifically bound to the template microtubules via specific linker molecules or the second microtubule binding site in their tail domain, respectively (step II). For both approaches, based on either biotemplated stamping or binding, the addition of “transport” microtubules in a motility solution containing ATP led to guided movement along the motor tracks (step III). Experiments on the microtubule-assisted stamping and binding of motor proteins were performed in 2-mm-wide flow cells self-built from two coverslips (Corning, 22 × 22 mm 2 and 18 × 18 mm 2 ) and two pieces of double-sided sticking tape (Scotch 3M, thickness 0.1 mm). Microtubules were polymerized from 5 μL of bovine brain tubulin (4 mg/ mL; labeled with different fluorophores as stated below) in BRB80 buffer (80 mM potassium PIPES, pH 6.9, 1 mM EGTA, 1 mM MgCl 2 ) with 4 mM MgCl 2 , 1 mM Mg-GTP, and 5% DMSO at 37 °C. After 30 min, the microtubule polymers were stabilized and diluted 100-fold in room- temperature BRB80 containing 10 μM taxol. Fluorescent images were obtained using a Zeiss Axiovert 200M inverted * Corresponding author. E-mail: diez@mpi-cbg.de. ² Max-Planck-Institute of Molecular Cell Biology and Genetics. ‡ The Nencki Institute of Experimental Biology. § University of Florida. NANO LETTERS 2006 Vol. 6, No. 10 2177-2183 10.1021/nl060922l CCC: $33.50 © 2006 American Chemical Society Published on Web 09/01/2006