Molecular robots guided by prescriptive landscapes

Abstract

Traditional robots rely for their function on computing, to store internal representations of their goals and environment and to coordinate sensing and any actuation of components required in response. Moving robotics to the single-molecule level is possible in principle, but requires facing the limited ability of individual molecules to store complex information and programs. One strategy to overcome this problem is to use systems that can obtain complex behaviour from the interaction of simple robots with their environment. A first step in this direction was the development of DNA walkers, which have developed from being non-autonomous to being capable of directed but brief motion on one-dimensional tracks. Here we demonstrate that previously developed random walkers-so-called molecular spiders that comprise a streptavidin molecule as an inert 'body' and three deoxyribozymes as catalytic 'legs'-show elementary robotic behaviour when interacting with a precisely defined environment. Single-molecule microscopy observations confirm that such walkers achieve directional movement by sensing and modifying tracks of substrate molecules laid out on a two-dimensional DNA origami landscape. When using appropriately designed DNA origami, the molecular spiders autonomously carry out sequences of actions such as 'start', 'follow', 'turn' and 'stop'. We anticipate that this strategy will result in more complex robotic behaviour at the molecular level if additional control mechanisms are incorporated. One example might be interactions between multiple molecular robots leading to collective behaviour; another might be the ability to read and transform secondary cues on the DNA origami landscape as a means of implementing Turing-universal algorithmic behaviour.

Figure 1. Deoxyribozyme based molecular walker and origami prescriptive landscape schematics

a, The NICK3.4A₃₊₁ spider consists of a streptavidin core that displays a 20 base ssDNA that positions the spider at the start (green), and three deoxyribozyme legs. b, The 8-17 deoxyribozyme cleaves its substrate at an RNA base creating two shorter products (seven and eleven bases). Dissociation from these products allows legs to associate with the next substrate. c, Spider actions: after release by a 27-base ssDNA trigger, the spider follows the substrate track, turns, and continues to a stop site (red). d, Schematic of the DNA origami landscape with positions A-E labeled; track EABD is shown. e, A representative origami landscape shows the start position (green), the substrate track (brown), stop and control sites (red), and a topographical marker (blue),

Figure 2. Results of spider movement along three tracks with schematics and AFM images of the spider at the start, on the track, and at the stop site

a, ABD track. b, EABC track. c, Graph of ABD and EABC spider statistics before and 30 minutes after release. d, EABD track. e, EABD track with spider on control. f, EABD product-only track. g, Graph of the EABD spider statistics before, and 15, 30 and 60 min after release, and 60 min after release on the EABD product-only track. All AFM images are 144 x 99.7 nm, the scale bar is 20 nm. Legend text indicates the number of origami with a single spider that were counted for the given sample.

Figure 3. AFM movie of spider movement

a, b, c, d, Schematics and AFM images of the spider moving along the EABD track at 5 min (a), 16 min (b), 26 min, (c) and 31 min (d) after trigger was added. AFM images are 300 × 300 nm and the scale bar is 100 nm.

Figure 4. Spiders imaged on origami tracks in real-time using super-resolution TIRF microscopy

a, Position-time trajectory of a selected spider (EAC 2, Cy3-labeled) on the EAC substrate track. The position as a function of time is represented by color-coded dots (see Supplementary Information for details). A small green dot represents the START and a large red oval represents the Cy5-labeled STOP site. ZnSO₄ was added at time zero. b, Displacement of the spider trajectory in panel a from its initial position as a function of time. The green line represents displacement calculated using averaged position measurements of 1 min intervals, and the black line represents the displacement from a rolling 4-min average (see Supplementary Information). c, Ensemble root mean square displacement (RMSD) of exemplary spiders on the EAC substrate track in the presence (red, corresponding to the 15 Tier 1 Spiders in Supplementary Fig. 29) and absence (black, 7 spiders) of Zn²⁺, with the corresponding displacements used to calculate each ensemble RMSD for each buffer condition (similarly colored line graphs). d, Ensemble RMSD for spiders on EAC tracks satisfying simple filtering criteria. Curves are shown for spiders on EAC substrate track (red, 85 spiders), EAC product track with TRIGGER introduced to the sample 10-15 min before imaging (blue, 18 spiders), and EAC product track with TRIGGER introduced 30-60 min before imaging (black, 29 spiders). EAC substrate and 10-15 min trigger product RMSD plots are fit to a power law function, and the EAC 30-60 min trigger product RMSD is fit to a straight line. Individual displacements are shown with colors corresponding to the respective ensemble RMSD plots. All Figure 4 data were obtained in SSC buffer.