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Shih, William
Assistant Professor
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.
An outstanding problem in biology is the efficient structure determination of transmembrane proteins. Residual dipolar couplings (RDCs), commonly measured for biological macromolecules weakly aligned by liquid-crystalline media, are important global orientation restraints for NMR structure determination. However, none of the existing liquid-crystalline media used to align water-soluble proteins are compatible with the detergents required to solubilize membrane proteins. We generated detergent-resistant liquid crystals of 0.8-m-long DNA nanotubes that enable weak alignment of detergent-reconstituted ζ-ζ transmeambrane domain of the T-cell receptor. Measurements of backbone NH and CH RDCs validate the high-resolution structure of the transmembrane homodimer. This DNA-nanotube liquid crystal will extend the advantages of weak alignment to NMR structure determination of membrane proteins. We also are working to build 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.
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.
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.
References:
Douglas SM, Chou JJ, Shih WM. DNA-nanotube-induced alignment of membrane proteins for NMR structure determination. Proc. Natl. Acad. Sci. USA 104, 6644-6648, 2007.
PNAS commentary: Sanders CR. Visiting order on membrane proteins by using nanotechnology. Proc. Natl. Acad. Sci. USA 104, 6502-6503, 2007.
Shih WM, Quispe JD, Joyce GF. A 1.7-kilobase single-stranded DNA that folds into a nanoscale octahedron. Nature 427, 618-621, 2004.
Shih WM, Gryczynski Z, Lakowicz JR, Spudich JA. 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. « Back
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