Achieving spatiotemporal control over molecular interactions with biological material-based approaches.

摘要

Spatiotemporal control of biomolecules and molecular interactions is an important aspect of the application of nanotechnology to the biological realm. Nature has demonstrated the ability to create highly efficient reactions at small scales through approaches that use multidimensional-based constraints. Solution based and membrane based reactions are three- and two-dimensional in nature, respectively. Cells also have the ability to use one-dimensional constrained reactions through nucleic acid and cytoskeletal reactions. By using this small scale inspiration through the cytoskeleton in vitro, we propose a novel method for accelerating the rate of biochemical reactions by controlling the spatiotemporal reactions using synthetic templating. As a model system, we use tetrameric beta-galactosidase, split into a pair of non-reactive dimers, and cause them to have a strong affinity for microtubules. The microtubule templating of the beta-galactosidase increases the concentration of the dimers to a level at which they will frequently reform into an active tetramer. The reactive activity of the beta-galactosidase is monitored using X-Gal, a substrate that forms a blue dye upon cleavage by the enzyme, enabling us to demonstrate a nearly 100-fold increase in the initial rate of reaction.;The same cytoskeletal system that provides the basis for our templating approach is used by the cell for transportation of macromolecules and organelles as well. The kinesin/microtubule interaction has been investigated for some time as a biologically based system for nano-scale transport, primarily using the microtubules as shuttles to move over a field of surface-adsorbed kinesin. We describe here a method of spatiotemporal manipulation of this interaction in order to control gliding microtubule motility dynamically, using the thermoresponsive polymer poly(N-isopropylacrylamide) and a PDMS microchannel liquid heating system. With this system, we can shuttle our reaction scaffolds to specific sites in order to localize the reaction even further.