William M. Shih
Department of Cancer Biology
A pivotal challenge for nanotechnology in the next half-century is to achieve precise positional control of material on the 1–100 nanometer scale. In light of this challenge, our laboratory explores rational design and directed evolution approaches to developing self-assembling DNA structures and devices with application to problems of biomedical interest.
A key property of DNA — its ability to be amplified exponentially by polymerases — facilitates the large-scale clonal production of individual sequences. This property also makes possible the directed evolution of sequence lineages toward optimized behaviors. Previous examples of three-dimensional geometric DNA objects, however, were built using architectures that are not amenable to copying by polymerases. We have developed a strategy for encoding DNA cages as single strands that are amplifiable by polymerases and that can be folded into a target structure by a simple denaturation-renaturation procedure. Our demonstration of a clonable DNA octahedron represents a large step toward making the use of DNA scaffolds more practical and more versatile.
An outstanding problem in biology is the efficient structure determination of transmembrane proteins. We are working to evolve DNA scaffolds that rigidly position lipid-bilayer-embedded transmembrane proteins into two-dimensional crystalline arrays and stacked-bilayer, three-dimensional crystalline arrays for analysis using electron and x-ray diffraction methods.
We are interested in using directed DNA evolution to explore the origins of molecular motor functionality in a Darwinian context. We ask questions such as the following: How easily can molecular motors be evolved from pools that are populated by DNA molecules encoding (a) completely random sequences, (b) random sequence variants of a catalyst, or (c) random sequence variants of a ligand-activated mechanical switch? What mechanisms of molecular movement emerge given varying selection pressures? Molecular motors generated from these studies will be integrated with static DNA structures for nanorobotic applications.
Shih, W. M., Quispe, J. D. & Joyce, G. F. A 1.7-kilobase single-stranded DNA that folds into a nanoscale octahedron. Nature 427, 618–621 (2004).
Shih, W. M. & Spudich, J. A. The myosin relay helix to converter interface remains intact throughout the actomyosin ATPase cycle. J. Biol. Chem. 276, 19491–19494 (2001).
Shih, W. M., Gryczynski, Z., Lakowicz, J. R. & Spudich, J. A. A FRET-based sensor reveals ATP hydrolysis-induced large conformational changes and three distinct states of the molecular motor myosin. Cell 102, 683–694 (2000).
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