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. In collaboration with James Chou at Harvard Medical School, we generated detergent-resistant liquid crystals of 0.8-μm-long DNA nanotubes that enable weak alignment of detergent-reconstituted membrane proteins. This DNA-nanotube liquid crystal will introduce the advantages of weak alignment to NMR structure determination for a number of membrane proteins. To generalize further the method (e.g. compatibility with positively-charged protein-micelle complexes) and to facilitate measurement of linearly-independent restraints (i.e. more structural information), we are working to generate additional DNA-nanostructure-based alignment media. We are applying our DNA-nanotools towards structure determination and mechanistic analysis of mitochondrial membrane proteins and GPCRs.
Single-molecule approaches provide a powerful tool for mechanistic investigation of biomolecular systems. Compared to bulk methods, analysis of individual molecules allows more direct measurement of microscopic forces and a more direct observation of changes in microscopic state, oftentimes obviating the need for technically challenging system synchronization. We seek to take advantage of the fine positional control afforded by DNA nanostructures to constrain and report on biomolecular complexes in ways that facilitate their study using single-molecule approaches. For example, exerting forces with nanoscale devices (as oppose to top-down devices such as optical traps or atomic-force microscopes) can be used to produce a system that in many cases is more amenable to study by single-molecule fluorescence.
DNA nanotechnology affords unprecedented control over macromolecular shape and site-specific functionalization. We are investigating the effect of DNA nanostructure shape, size, and chemical functionalization on the rate of uptake of such particles into cells. As a longer-term challenge, we also seek to design DNA-scaffolded molecular machines that enable controlled passage through biological membranes, by either triggered membrane fusion or triggered membrane pore formation. Another long-term challenge is to form 3D scaffolds with programmable structural properties that present bioactive cell-adhesive molecules, morphogens, and growth factors with defined spatiotemporal control and release kinetics so as to produce appropriate chemical gradients and micromechanical cues for directing effective tissue and organ self-assembly both in vitro and in vivo. DNA nanotechnology promises an unprecedented level of spatiotemporal control using a naturally biocompatible and biodegradable material that in principle could be exploited for meeting this challenge. As first steps in a collaboration with the laboratory of Don Ingber, we seek to demonstrate actuation potential and mechanical controllability of prestressed DNA tensegrities, to create DNA-protein chimeras that integrate defined 3D DNA nanostructures into natural extracellular matrices, and to use these artificial matrices to control mammalian cell behavior and multicellular organization by mechanically actuating physical changes in the internal DNA nanostructures.
The complexity of integrated circuits has doubled every eighteen months or so over the past 40 years. This staggering increase in computing capability has transformed society. Our field seeks a similar trajectory of exponential advancement for programmable self-assembling systems. We try to maintain subnanometer positional control while constructing DNA scaffolds that are ever larger and more complex. At the same time, we think about how we could scale up the mass quantities of scaffolds that we could produce, towards constructing macroscale objects from these materials. We also are interested in generating dynamic molecular machines and devices.
Liedl T, Högberg B, Tytell J, Ingber DE, Shih WM. Self-assembly of 3D prestressed tensegrity structures from DNA. Nature Nanotechnology in press, 2010.
Dietz H, Douglas SM, Shih WM. Folding DNA into twisted and curved nanoscale shapes. Science 325, 725–730, 2009.
Douglas SM, Dietz H, Liedl T, Högberg B, Graf F, Shih WM. Self-assembly of DNA into nanoscale three-dimensional shapes. Nature 459, 414–418, 2009.
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.