Traditional robots 1 rely on computing to coordinate sensing and actuating components and to store internal representations of their goals and environment. Any implementation of single-molecule based robotics must overcome the limited ability of individual molecules to store complex programs and, for example, use architectures that obtain complex behaviors from the interaction of simple robots with their environment [2][3][4] . Previous research in DNA walkers 5 focused on transitioning from non-autonomous systems 6, 7 to directed but brief motion on one-dimensional tracks8 -11. Herein, we obtain elementary robotic behaviors from the interaction between a random walker incorporating deoxyribozymes 12 and a precisely defined environment. Using singlemolecule microscopies we demonstrate that such walkers achieve directionality by sensing and modifying their environment, following trails of recognition elements ("bread crumbs") laid out on a two-dimensional DNA origami landscape 13 . These molecular robots autonomously carry out sequences of actions such as "start", "follow", "turn", and "stop", thus laying the foundation for the synthesis of more complex robotic behaviors at the molecular level by incorporating additional layers of control mechanisms. For example, interactions between multiple molecular robots could lead to collective behavior14 , 15, while the ability to read and transform secondary cues on the landscape could provide a mechanism for Turing-universal algorithmic behavior2 ,16,17 .Author Contributions: AFM experiments were performed by K.L. (majority), J.N., and N. D.; analysis was performed by N. D., K.L., J.N., S.T., and supervised by E.W., and H.Y. Fluorescence microscopy and particle tracking analysis were performed by A.J.M., N.M., and A.J.B, supervised by N. G. W. Spiders were synthesized, purified, and their integrity confirmed and monitored by S.T. SPR experiments were performed by R. P. Research coordination by M.N.S., material transfer coordination by S.T., J.N., and K.L. Experimental design and manuscript was done with input from all authors. Author Information: Reprints and permissions information is available at npg.nature.com/reprintsandpermissions. The authors declare no competing financial interests. Correspondence and requests for materials should be addressed to: mns18@columbia.edu, winfree@caltech.edu, nwalter@umich.edu, hao.yan@asu.edu Supplementary Information is linked to the online version of the paper at www.nature.com/nature. NIH Public Access Author ManuscriptNature. Author manuscript; available in PMC 2010 November 1. Restated in a biochemically more intuitive manner: A deoxyribozyme on a site that was previously converted to a product will dissociate faster, whereas it will stick longer on the substrates and eventually cleave them. Because spiders have multiple legs, a single dissociated leg will quickly reattach to nearby product or substrate. It follows that the body of a spider positioned at the interface between products and substrates will move toward the s...

We have studied the correlation between structural dynamics and function of the hairpin ribozyme. The enzyme-substrate complex exists in either docked (active) or undocked (inactive) conformations. Using single-molecule fluorescence methods, we found complex structural dynamics with four docked states of distinct stabilities and a strong memory effect where each molecule rarely switches between different docked states. We also found substrate cleavage to be rate-limited by a combination of conformational transitions and reversible chemistry equilibrium. The complex structural dynamics quantitatively explain the heterogeneous cleavage kinetics common to many catalytic RNAs. The intimate coupling of structural dynamics and function is likely a general phenomenon for RNA.

The general design of the swinging arm nanostructure complex is shown in Fig. 1a, where a two-enzyme cascade consisting of glucose-6 phosphate dehydrogenase (G6pDH) 19 and malic dehydrogenase (MDH) 20 is displayed on a DNA double-crossover (DX) tile scaffold 21 (DNA sequences for all structures used in this study are shown in Supplementary Figs. S1-S7).G6pDH catalyzes the oxidation of glucose-6-phosphate and the reduction of NAD + to NADH.Subsequently, MDH catalyzes the reduction of oxaloacetate to malic acid using the NADH produced by G6pDH. To facilitate channeling of NADH between G6pDH and MDH, an NAD + -3 functionalized poly(T) 20 oligonucleotide was attached to the DNA tile surface halfway between G6pDH and MDH (see Supplementary Figs. S8-S21 for a detailed description of conjugation, assembly and purification of the nanostructured complex). Fig. 1b shows a native polyacrylamide gel electrophoresis (PAGE) analysis of the assembled enzyme complex, together with various sub-complexes. Both the gel results and the chromatogram resulting from sizeexclusion chromatography ( Supplementary Fig. S21) demonstrate assembly of the G6pDH-NAD + -MDH swinging arm cascade with >80% yield. The assembled mixture was further purified by size exclusion chromatography for enzyme activity assays. Assembly of the complete complex was also visualized by atomic force microscopy (AFM) ( Fig. 1c and Supplementary Fig. S57 for larger view images), where the presence of the enzymes on the structure is confirmed by differences in height ("brightness") compared to the surface of the DNA tile.To first explore the parameters and understand the kinetics and mechanism of the restricted diffusion mediated by the ssDNA swinging arm, we developed a simplified model system ( Supplementary Fig. S22). In this model, a Cy3 reporter dye takes the place of NAD + on the single-stranded poly(T) 20 arm, whereas a BHQ fluorescence quencher and a Cy5 energy transfer acceptor dye replace one or both enzymes on selected probe positions surrounding the swinging arm (Fig. 2a). An oligonucleotide sequence (5'-ATA GTG AAA) was extended from the 5' end of the poly(T) 20 sequence and positioned halfway between the quencher and acceptor, allowing the arm to transiently hybridize to the probes that each bear the complementary sequence (5'-TTT CAC TAT) in analogy to the binding of NAD + /NADH to the dehydrogenases. To characterize the distance dependence of the diffusive transport and binding mediated by the swinging arm using smFRET, we chose a design in which a single Cy5-labeled capture probe was placed at one of three topologically accessible distances from the Cy3-labeled arm: 7 nm (21 base pairs), 14 nm (42 base pairs) and 21 nm (63 base pairs). As shown in Fig. 2b, the most efficient hybridization to the capture probe was observed at 7 nm, where ~94% of all swinging arms were associated with the Cy5 probe at equilibrium (leading to high FRET). As the distance increased, the equilibrium fraction of captured swinging arms decreased to ~58% at 14 nm and only ...