Imaging an Expanding Molecular Robot World Using Super-Accuracy Single-Molecule Fluorescence Microscopy

We recently demonstrated the concept of molecular robotics with a synthetic DNA-based nanowalker, dubbed a “spider” composed of a streptavidin protein “body” attached to three biotinylated DNA enzyme legs, along a one-dimensional track of chimeric DNA-RNA substrates positioned on a DNA origami (1,2). By cleaving its substrates, the spider weakens the binding energy between its legs and previously visited sites, resulting in a biased, processive, random walk towards fresh substrate. Additional components are now being incorporated into the spider world to increase its versatility and complexity in behavior. For example, we are implementing a second spider that walks on a different substrate. This spider can be differentially controlled from our original nanowalker via changes in buffer conditions that favor one spider over the other and vice versa. This advance will allow us to devise a controlled “spider race” towards a common goal post; depending on the predetermined winner, a payload will either be released from the goal or not, behaving as an XOR gate. By fluorophore labeling the different components, we aim to characterize this spider race at the single molecule level using super-accuracy total internal reflection fluorescence microscopy (TIRFM).1. Lund, K., Manzo, A.J., Dabby, N., Michelotti, N., Johnson-Buck, A., Nangreave, J., Taylor, S., Pei, R., Stojanovic, M.N., Walter, N.G., Winfree, E., and Yan, H. (2010) Molecular robots guided by prescriptive landscapes. Nature 465, pp. 206–210.2. Michelotti, N., de Silva, C., Johnson-Buck, A.E., Manzo, A.J., Walter, N.G. (2010) A bird's eye view: tracking slow nanometer-scale movements of single molecular nano-assemblies. Methods Enzymol. 475, pp. 121–148.