Peng Yin, Harry M. T. Choi, Colby R. Calvert and Niles A. Pierce - Programming Biomolecular Self-Assembly Pathways

Vol 451 | 17 January 2008 | doi:10.
1038/nature06451
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Programming biomolecular self-assembly pathways
Peng Yin1,2, Harry M. T. Choi1, Colby R. Calvert1 & Niles A. Pierce1,3
In nature, self-assembling and disassembling complexes of proteins and nucleic acids bound to a variety of ligands perform intricate and diverse dynamic functions. In contrast, attempts to rationally encode structure and function into synthetic amino acid and nucleic acid sequences have largely focused on engineering molecules that self-assemble into prescribed target structures, rather than on engineering transient system dynamics1,2. To design systems that perform dynamic functions without human intervention, it is necessary to encode within the biopolymer sequences the reaction pathways by which self-assembly occurs. Nucleic acids show promise as a design medium for engineering dynamic functions, including catalytic hybridization36, triggered self-assembly7 and molecular computation8,9. Here, we program diverse molecular self-assembly and disassembly pathways using a reaction graph abstraction to specify complementarity relationships between modular domains in a versatile DNA hairpin motif. Molecular programs are executed for a variety of dynamic functions: catalytic formation of branched junctions, autocatalytic duplex formation by a cross-catalytic circuit, nucleated dendritic growth of a binary molecular tree, and autonomous locomotion of a bipedal walker. The hairpin motif (A in Fig. 1a) comprises three concatenated domains, a, b and c. Each domain contains a special nucleation site called a toehold10, denoted at, bt and ct. Two basic reactions can be programmed using this motif, as illustrated for the example of catalytic duplex formation in Fig. 1b. First, an assembly reaction (1) occurs when a single-stranded initiator I, containing an exposed toehold at*, nucleates at the exposed toehold at of hairpin A, initiating a branch migration that opens the hairpin. Hairpin domains b and c, with newly exposed toeholds bt and ct, can then serve as assembly initiators for other suitably defined hairpins, permitting cascading (for example, in reaction (2), domain b of hairpin A assembles with domain b* of hairpin B, opening the hairpin). Second, a disassembly reaction (3) occurs when a single-stranded domain (a* of B) initiates a branch migration that displaces the initiator I from A. In this example, I catalyses the formation of duplex A N B through a prescribed reaction pathway. To assist in programming more complex reaction pathways, we abstract the motif of Fig. 1a as a node with three ports (Fig. 1c): a triangular input port and two circular output ports. The state of each port is either accessible (open triangle/circle) or inaccessible (solid triangle/circle), depending on whether the toehold of the corresponding motif domain is exposed or sequestered. Functional relationships between ports within a node are implicit in the definition of the nodal abstraction corresponding to a particular motif (for example, for the node of Fig. 1c, the output ports flip to accessible states if the input port is flipped to an inaccessible state through an interaction with a complementary upstream output port). By depicting assembly reactions by solid arrows and disassembly reactions by dashed arrows (each directed from an output port to a complementary input port of a different node), reaction pathways can be specified abstractly in the form of a reaction graph, representing a program to be executed by nucleic acid molecules. The reactions depicted in the secondary structure mechanism of Fig. 1b are specified using a reaction graph in Fig. 1d. The initial conditions for this program are described via the state of each port in the reaction graph. Figure 1e depicts the execution of this reaction graph through cascaded assembly and disassembly reactions. An assembly reaction is executed when ports connected by a solid arrow are simultaneously accessible. For the initial conditions depicted in Fig. 1d, the program must start with the execution of reaction (1). Reaction 1 (assembly): in an assembly reaction (executed here by the accessible output port of I and the complementary accessible input port of A), a bond is made between the ports and they are flipped to inaccessible states; the two output ports of A are flipped
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Figure 1 | Programming biomolecular self-assembly pathways. a, Secondary structure of the hairpin motif. Coloured lines represent strand domains; short black lines represent base pairs; arrowheads indicate 39 ends. Domain c is optional. b, Secondary structure mechanism illustrating assembly and disassembly reactions during catalytic duplex formation. Asterisks denote complementarity. c, Abstraction of the motif A as a node with three ports (colour use is consistent with a). d, A reaction graph representing a molecular program executed schematically in b and e. e, Execution of the reaction graph of d. f, Hierarchical design process.
1 Department of Bioengineering, 2Department of Computer Science, 3Department of Applied & Computational Mathematics, California Institute of Technology, Pasadena, California 91125, USA.
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to accessible states (based on the internal logic of node A). Reaction 2 (assembly): a bond is made between the newly accessible blue output port of A and the complementary accessible input port of B and both ports are flipped to inaccessible states; the output port of B is flipped to the accessible state (based on the internal logic of node B). Reaction 3 (disassembly): in a disassembly reaction (executed here by the newly accessible output port of B, the inaccessible input port of A, and the inaccessible output port of I), the bond between the output port of I and the input port of A is displaced by a bond between the output port of B and the input port of A; the states of the two output ports are flipped (see Supplementary Information 2 for additional details). The reaction graph provides a simple representation of assembly (and disassembly) pathways that can be translated directly into molecular executables: nodes represent motifs, ports represent domains, states describe accessibility, arrows represent assembly and disassembly reactions between complementary ports. Starting from a conceptual dynamic function, a molecular implementation is realized in three steps (Fig. 1f): (1) pathway specification via a reaction graph; (2) translation into secondary structure motifs; (3) computational design of motif primary sequences (see Methods for details). We demonstrate the utility of this hierarchical design process by experimentally executing molecular programs encoding four distinct dynamic functions. Program 1: Catalytic geometry. Current protocols for selfassembling synthetic DNA nanostructures often rely on annealing procedures to bring interacting DNA strands to equilibrium on the free-energy landscape1113. By contrast, self-assembly in biology proceeds isothermally and assembly kinetics are often controlled by catalysts. Until now, synthetic DNA catalysts36 have been used to control the kinetics of the formation of DNA duplex structures. The next challenge is to catalyse the formation of branched DNA structures, the basic building blocks for DNA structural nanotechnology14,15. First, we demonstrate the catalytic formation of a three-arm DNA junction. The assembly and disassembly pathways specified in the reaction graph of Fig. 2a are translated into the motif-based molecular implementation of Fig. 2b (see Supplementary Information 3.1 for details). The complementarity relationships between the segments of hairpins A, B, and C are specified (Fig. 2b, top) so that in the absence of initiator strand I, the hairpins are kinetically impeded from forming the three-arm junction that is predicted to dominate at equilibrium. In the reaction graph, this property is programmed by the absence of a starting point if node I is removed from the graph (that is, no pair of accessible ports connected by an assembly arrow). The introduction of I into the system (Fig. 2b, bottom) activates a cascade of assembly steps with A, B and C, followed by a disassembly
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step in which C displaces I from the complex, freeing I to catalyse the self-assembly of additional branched junctions. Gel electrophoresis confirms that the hairpins assemble slowly in the absence of initiator and that assembly is markedly accelerated by the addition of initiator (Fig. 2c). Disassembly of the initiator leads to catalytic turnover, as indicated by the nearly complete consumption of hairpins even at substoichiometric initiator concentrations. Interestingly, only minimal assembly is achieved by annealing the hairpin mixture, illustrating the utility of pathway programming for traversing free-energy landscapes with kinetic traps that cannot be overcome by traditional annealing approaches. Direct imaging of the catalysed self-assembly product A N B N C by atomic force microscopy (AFM) reveals the expected three-arm junction morphology (Fig. 2d). In principle, the reaction pathway can be extended to the catalytic self-assembly of k-arm junctions (Supplementary Information 3.5). We illustrate k 5 4 with the reaction graph and AFM image of Fig. 2e and f. Program 2: Catalytic circuitry. By programming cross-catalytic self-assembly pathways in the reaction graph of Fig. 3a, we obtain an autocatalytic system with exponential kinetics. In the corresponding molecular implementation, four hairpin species, A, B, C and D, coexist metastably in the absence of initiator I (Fig. 3b, top). The initiator catalyses the assembly of hairpins A and B to form duplex A N B (steps 12, Fig. 3b, bottom), bringing the system to an exponential amplification stage powered by a cross-catalytic circuit: the duplex A N B has a single-stranded region that catalyses the assembly of C and D to form C N D (steps 34); duplex C N D in turn has a singlestranded region that is identical to I and can thus catalyse A and B to form A N B (steps 56). Hence, A N B and C N D form an autocatalytic set capable of catalysing its own production. Disassembly (steps 2b, 4b and 6b) is fundamental to the implementation of autocatalysis and sterically uninhibited exponential growth. Each step in the reaction is examined using native polyacrylamide gel electrophoresis (Supplementary Fig. 12), showing the expected assembly and disassembly behaviour. System kinetics are examined in a fluorescence quenching experiment (Fig. 3c). Spontaneous initiation in the absence of initiator reflects the finite timescale associated with the metastability of the hairpins and yields a sigmoidal time course characteristic of an autocatalytic system16. As expected, the curve shifts to the left as the concentration of initiator is increased. A plot of 10% completion time against the logarithm of the concentration shows a linear regime, consistent with exponential kinetics and analytical modelling (Fig. 3c, inset). The minimal leakage of a system containing only A and B (labelled A 1 B in Fig. 3c) emphasizes that the sigmoidal kinetics of spontaneous initiation for the full system (A 1 B 1 C 1 D) are due to cross-catalysis.
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Figure 2 | Programming catalytic geometry: catalytic self-assembly of three-arm and four-arm branched junctions. See Supplementary Information 3 for details. a, Reaction graph for three-arm junctions. b, Secondary structure mechanism. Each letter-labelled segment is six nucleotides in length. The initially accessible (a* for step 1) or newly exposed (b* for Step 2, c* for step 3) toeholds that mediate assembly reactions are labelled with purple letters. c, Agarose gel electrophoresis demonstrating
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Figure 3 | Programming catalytic circuitry: autocatalytic duplex formation by a cross-catalytic circuit with exponential kinetics. See Supplementary Information 4 for details. a, Reaction graph. Multiple assembly arrows entering the same input port depict parallel processes on separate copies of the nodal species. b, Secondary structure mechanism. c, System kinetics examined by fluorescence quenching. Formation of A N B is monitored by the increase in fluorescence resulting from increased spatial separation between the fluorophore (green star in b) and the quencher (black dot in b) at either
end of A. Raw data for two independent reactions are displayed for each initiator concentration (20-nM hairpins). Single traces are shown for the controls containing only A and B or only A. Inset: linear fit of the 10% completion time against the logarithm of the relative concentration of I (0.0033 # [I] # 0.053). High-concentration end points ([I] $ 0.13) are excluded based on theoretical analysis; low-concentration end points ([I] # 0.0013) are excluded because of signal poisoning by leakage. See Supplementary Information 4.4 for a detailed treatment.
This system demonstrates synthetic biomolecular autocatalysis1720 driven by the free energy of base-pair formation. Autocatalysis and exponential system kinetics can also be achieved through entropydriven hybridization mechanisms21. For sensing applications, the triggered exponential growth of these systems suggest the possibility of engineering enzyme-free isothermal detection methods. Program 3: Nucleated dendritic growth. The molecular program in Fig. 4a depicts the triggered self-assembly of a binary molecular tree of a prescribed size. The reaction starts with the assembly of an
initiator node I with a root node A1. Each assembled node subsequently assembles with two child nodes during the next generation of growth, requiring two new node species per generation. In the absence of steric effects, a G-generation dendrimer requires 2G 1 node species and yields a binary tree containing 2G1 monomers, that is, a linear increase in the number of node species yields an exponential increase in the size of the dendrimer product. Figure 4b depicts the motif based implementation of the program depicted in Fig. 4a: hairpins are metastable in the absence of initiator; the
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Figure 4 | Programming nucleated dendritic growth: triggered assembly of quantized binary molecular trees. See Supplementary Information 5 for details. a, Reaction graph. Multiple assembly arrows entering the same input port depict parallel processes on separate copies of the nodal species. b, Secondary structure mechanism. c, Agarose gel electrophoresis demonstrating triggered self-assembly. Lanes 16: the dominant reaction band shifts with the addition of each generation of hairpins. Subdominant
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bands are presumed to represent imperfect dendrimers. Lane 7: minimal conversion to reaction products in the absence of initiator. Hairpins A1, A2, B2 at 62.5 nM; the concentration doubles for each subsequent generation of hairpins. Initiator I at 50 nM. d, Linear relationship between amplification signal (putative G5 reaction product) and initiator for three independent experiments (cross, diamond, circle). See Supplementary Fig. 17 for details. e, AFM images of G3, G4 and G5 dendrimers. Scale bars: 30 nm.
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initiator I triggers the growth of a dendrimer with five generations of branching (G5). We constructed trees with G 5 1, 2, 3, 4 and 5. The nucleated growth of the tree is examined using native agarose gel electrophoresis. Band shifting demonstrates increasing dendrimer size with each generation of growth (Fig. 4c). Figure 4d demonstrates that the concentration of dendrimer depends linearly on the concentration of the initiator in the system. Finally, AFM imaging of dendrimers for G 5 3, 4 and 5 reveals the expected morphologies (Fig. 4e). Measurements of the dendrimer segment lengths agree well with the design (Supplementary Information 5.4). In contrast to previous work in which DNA dendrimer target structures were synthesized by sequential ligation of structural subunits22, here we program self-assembly pathways so that DNA monomers form dendrimers only on detection of a target nucleation molecule. By growing to a prescribed size, these dendrimers provide quantitative signal amplification with strength exponential in the number of constituent species. Program 4: Autonomous locomotion. The challenge of engineering molecular machines capable of nanoscale autonomous locomotion has attracted much interest in recent years2327. Inspired by
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Figure 5 | Programming autonomous locomotion: stochastic movement of a bipedal walker. See Supplementary Information 6 for details. a, Reaction graph. Bonds between output ports on I and input ports on A represent initial conditions. Static structural elements are depicted by grey line segments. b, Secondary structure mechanism depicting processive locomotion. See Supplementary Information 6.1 and 6.3 for non-processive trajectories. cf, Fluorescence quenching experiments measuring the proximity of the quenchers (black dots) on the walker feet to the fluorophores (coloured stars) decorating the track. Fitted curves (solid) are used to determine the time at which the minimum fluorescence (maximum quenching) was observed (dashed vertical line) for each fluorophore. c, Bipedal walker with track labelled by fluorophores JOE (green star) R TAMRA (red) R FAM (blue) as in b. For each pair of consecutive minima (JOE R TAMRA and TAMRA R FAM), we test the null hypothesis that the median time difference between the minima is zero against the alternative hypothesis that the time difference is positive. Based on a statistical analysis of six independent experiments (see Supplementary Information 6.6, 6.7), the null hypothesis can be rejected for both time differences with the same P-value of 0.0156, supporting the interpretation that the observed minima are sampled from a distribution in which the ordering of the minima matches the physical ordering of the fluorophores along the track. Similar interpretations apply to the ordering of minima for d and f. d, Monopedal walkers on the same track (JOE (orange star) R TAMRA (pale green) R FAM (pale blue)). e, Comparison of time scales for bipedal and monopedal walkers (eighteen traces per walker type: three fluorophores, six experiments). f, Bipedal walker with track labelled TAMRA (red star) R JOE (green) R FAM (blue).
the bipedal motor protein, kinesin, which hauls intracellular cargo by striding along microtubules28, we have developed an autonomous enzyme-free bipedal DNA walker capable of stochastic locomotion along a DNA track. Joined by a duplex torso, each of two identical walker legs, I, is capable of catalysing the formation of waste duplex A N B from metastable fuel hairpins A and B through a reaction pathway in which I assembles with A, which assembles with B, which subsequently disassembles I from the complex (see Fig. 5a and b for the reaction graph and corresponding molecular implementation). The track consists of five A hairpins arranged linearly at regular intervals along a nicked DNA duplex. In the presence of hairpin B, a subpopulation of walkers is expected to move unidirectionally along the track by sequentially catalysing the formation of A N B. Because of the one-dimensional arrangement of anchor sites, this processive motion occurs only for those walkers that use a foot-over-foot gait by stochastically lifting the back foot at each step. We investigate walker locomotion using a bulk fluorescence assay that tests whether there is a subpopulation of walkers that moves processively through positions 3, 4 and 5, starting from an initial condition with legs anchored at positions 1 and 2. Quenchers are attached to the walkers legs and spectrally distinct fluorophores are positioned proximal to anchorages 3, 4 and 5. Consistent with processivity, the anticipated sequential transient quenching of the fluorophores at positions 3, 4 and 5 is observed (Fig. 5c). To rule out the possibility that this signal arises from non-processive walker diffusion through the bulk solution from one position to the next, we repeated the experiments using monopedal walkers that lack a mechanism for achieving processivity. In this case, the sequential transient quenching no longer matches the ordering of the fluorophores along the track (Fig. 5d) and the timescale for visiting any one of the three anchorages is longer than the timescale to visit all three anchorages for the bipedal system (Fig. 5e). Additional control experiments (Supplementary Information 6.9) show that this difference in timescales cannot be explained by the relative rates with which freely diffusing bipedal and monopedal walkers land on the track. As a further test of processivity for the bipedal walker, reordering the fluorophores along the track leads to the expected change in the ordering of the transient quenching (Fig. 5f). The experimental execution of these four molecular programs demonstrates that the hairpin motif functions as a modular programmable kinetic trap, and that rewiring the connections between nodes in the reaction graph corresponds to rewiring the connections between kinetic traps in the underlying free-energy landscape. In the physical systems, metastable hairpins are initially caught in engineered kinetic traps; the introduction of initiator molecules begins a chain reaction of kinetic escapes in which the hairpin species interact through programmed assembly and disassembly steps to implement dynamic functions. It is important that the timescale of metastability for kinetically trapped molecules is longer than the timescale relevant for the execution of the program. We found it helpful to incorporate clamping segments at the ends of helices to discourage the initiation of non-toehold-mediated branch migrations (see Supplementary Information 3.1). We also found that impure strand syntheses artificially reduce the strength of metastable traps and increase leakage rates. System fidelity was improved by ligating hairpins out of two shorter segments to increase strand purity (Supplementary Information 7.1). Reaction graphs can be extended beyond the present versatile motif by defining new nodal species that abstract the functional relationships between domains in other motifs. The present hierarchical approach to encoding dynamic function in nucleic acid sequences represents a promising step towards the goal of constructing a compiler for biomolecular functionan automated design process that requires as input a modular conceptual system design, and provides as output a set of biopolymer sequences that encode the
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desired dynamic system behaviour (Supplementary Information 7.2).

METHODS SUMMARY
Starting from a conceptual dynamic function, a molecular implementation is realized in three steps summarized in Fig. 1f. See Supplementary Information 3.1 for an example illustrating the design of the catalytic three-arm junction system. Step (1): pathway specification. We specify the pathway that implements a target dynamic function using a reaction graph. Step (2): translation to motifs. The reaction graph is directly translated to motif secondary structures. First, the basic complementarity requirements are defined and then clamping/padding segments are added (as in Supplementary Information 3.1). Initial dimensioning of the number of nucleotides in each segment is performed using the NUPACK server (www.nupack.org), which models the behaviour of strand species in the context of a dilute solution (including unintended species of complexes)29. Step (3): sequence design. Sequences are designed by considering a suite of structures that punctuate the intended reaction pathway or that explicitly preclude undesired off-pathway interactions (for example, structures specifying the absence of an interaction between two strands that should not pair). The sequences are optimized computationally (J. N. Zadeh and R. M. Dirks, personal communication) to maximize affinity and specificity for this suite of structures by minimizing the average number of incorrectly paired bases at equilibrium30. We then synthesize and verify the system using gel electrophoresis, bulk fluorescence quenching, or single-molecule AFM.
Full Methods and any associated references are available in the online version of the paper at www.nature.com/nature. Received 20 July; accepted 31 October 2007.
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Supplementary Information is linked to the online version of the paper at www.nature.com/nature. Acknowledgements We thank the following for discussions: J. S. Bois, R. M. Dirks, M. Grazier GSell, R. F. Hariadi, J. A. Othmer, J. E. Padilla, P. W. K. Rothemund, T. Schneider, R. Schulman, M. Schwarzkopf, G. Seelig, D. Sprinzak, S. Venkataraman, E. Winfree, J. N. Zadeh and D. Y. Zhang. We also thank J. N. Zadeh, R. M. Dirks and J. M. Schaeffer for the use of unpublished software, and R. F. Hariadi and S. H. Park for advice on AFM imaging. This work is funded by the NIH, the NSF, the Caltech Center for Biological Circuit Design, the Beckman Institute at Caltech, and the Gates Grubstake Fund at Caltech. Author Information Reprints and permissions information is available at www.nature.com/reprints. The authors declare competing financial interests: details accompany the paper on Natures website (http://www.nature.com/ nature). Correspondence and requests for materials should be addressed to N.A.P. (niles@caltech.edu).
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METHODS
System design. A molecular implementation is realized in three steps summarized in Fig. 1f and illustrated in Supplementary Information 3.1. Step (1): pathway specification. Step (2): translation to motifs. Following initial dimensioning using the NUPACK server, the segment dimensions are sometimes further optimized based on subsequent experimental testing. Step (3): sequence design. After computational optimization, occasional further manual optimization was performed using the same design metric on a subset of crucial target structures. We then further analysed the thermodynamic behaviour of the sequences using the NUPACK server. For some systems, stochastic kinetic simulations31 (J. M. Schaeffer, personal communication) were carried out to confirm the absence of significant kinetic traps along the target reaction pathways. The sequences are shown in Supplementary Information 8. System synthesis. DNA was synthesized and purified by Integrated DNA Technologies. The purified DNA strands were reconstituted in ultrapure water (resistance of 18 MV cm). We determined the concentrations of the DNA solutions by measuring ultraviolet light absorption at 260 nm. Hairpins were synthesized as two pieces which were then ligated to produce the full hairpin (see Supplementary Information 7.1 for details). We performed the ligation using T4 DNA ligase (New England Biolabs) at either room temperature or 16 uC for a minimum of 2 h. We further purified ligated strands using denaturing polyacrylamide gel electrophoresis. The bands corresponding to the DNA strands of expected sizes were visualized by ultraviolet shadowing and excised from the gel. The DNA strands were then eluted and recovered by ethanol precipitation. For monomer preparation, we diluted the concentrated DNA strands to reaction conditions: 50 mM Na2HPO4, 0.5 M NaCl, pH 5 6.8 for species in Fig. 2 and Supplementary Fig. 4; and 20 mM Tris, pH 5 7.6, 2 mM EDTA, 12.5 mM Mg21 (1 3 TAE/Mg21 buffer) for species in Fig. 3, Supplementary Fig. 12, and Fig. 4. We then annealed the hairpins by heating for 5 min at 90 uC, and then turning off the heating block to allow the system to cool to room temperature (requiring at least 2 h). For walker system assembly, see Supplementary Information 6.4. Gel electrophoresis. For the gel in Fig. 2c, 12 ml of each 3-mM hairpin species were mixed by pipetting. Portions of this master mix were aliquoted into five separate tubes (6 ml per tube). To these tubes we added 2 ml of either 3 mM I (lane 1), 1.5 mM I (lane 2), 0.75 mM I (lane 3), 0.3 mM I (lane 4), or 13 reaction buffer (50 mM Na2HPO4, 0.5 M NaCl, pH 5 6.8) (lane 5) to reach a total reaction volume of 8 ml. The samples were then mixed by pipetting and allowed to react for 2.5 h at room temperature. The annealed reaction (lane 6), prepared 0.5 h in advance, was made by mixing 2 ml of each hairpin with 2 ml of the 13 reaction buffer, and then annealing as described in monomer preparation. A 2% native agarose gel was prepared for use in 13 LB buffer (Faster Better Media, LLC). We then mixed 1 ml of each sample with 1 ml of 53 SYBR Gold loading buffer: 50% glycerol/50% H2O/SYBR Gold (Invitrogen) and loaded this into the gel. The gel was run at 350 V for 10 min at room temperature and imaged using an FLA-5100 imaging system (Fuji Photo Film). For the gel in Fig. 4c, we annealed the hairpins at the following concentrations: A1, A2, B2, A3 and B3 at 1 mM; A4 and B4 at 2 mM; A5 and B5 at 4 mM. The initiator I was prepared at 800 nM. The following sample mixtures were prepared: lane 1, A1; lane 2, I 1 A1; lane 3, I 1 A1 1 A2 1 B2; lane 4, I 1 A1 1 A2 1 B2 1 A3 1 B3; lane 5, I 1 A1 1 A2 1 B2 1 A3 1 B3 1 A4 1 B4; lane 6, I 1 A1 1 A2 1 B2 1 A3 1 B3 1 A4 1 B4 1 A5 1 B5; lane 7, A1 1 A2 1 B2 1 A3 1 B3 1 A4 1 B4 1 A5 1 B5. Here, I, A1, A2 and B2 were added at 1 ml; A3, B3, A4, B4, A5 and B5 at 2 ml. We added 13 reaction buffer (20 mM Tris, pH 5 7.6, 2 mM EDTA, 12.5 mM Mg21) to bring the total volume of each sample to 16 ml. We mixed the samples by pipetting and allowed them to react for 2 h at room temperature. A 1% native agarose gel was prepared in 13 LB buffer. We added 8 ml of each sample to 2 ml 53 SYBR Gold loading buffer and loaded 8 ml of this sample/loading-buffer mix into the gel. The gel was run at 350 V for 10 min at room temperature and then imaged using an FLA-5100 imaging system. For the reactions in Fig. 4d, the hairpins were mixed to reach the following final concentration: A1-Cy5 (see Supplementary Information 8.4),
A2, B2, 100 nM; A3, B3, 200 nM; A4, B4, 400 nM; A5, B5, 800 nM. We then aliquoted portions of this mix into 10 separate tubes (9 ml per tube). To these tubes we added either 13 TAE/Mg21 reaction buffer or the initiator I to give the indicated final concentration of I and a final volume of 11 ml. The samples were mixed by pipetting and allowed to react for 1 h at room temperature. We then mixed the sample with 53 LB loading buffer (Faster Better Media, LLC) to reach 13 loading buffer concentration (8 ml sample, 2 ml loading buffer). We loaded the sample/loading buffer mix into a 1% native agarose gel prepared in 13 LB buffer. The gel was run at 350 V for 10 min at room temperature and then imaged and quantified using an FLA-5100 imaging system. The experiments were performed with 10 mM inert 25-nt poly-T carrier strands21 in the reaction solution. AFM imaging. We obtained AFM images using a multimode scanning probe microscope (Veeco Instruments), equipped with a Q-Control module for analogue AFM systems (Atomic Force F&E). The images were obtained in liquid phase under tapping mode using DNP-S oxide sharpened silicon nitride cantilevers (Veeco). We first diluted samples in 13 TAE/Mg21 buffer to achieve the desired imaging density. We applied a 20 ml drop of 13 TAE/Mg21 and a 5 ml drop of sample to the surface of freshly cleaved mica and allowed them to bind for approximately 2 min. We added supplemental Ni21 (1530 mM) to increase the strength of DNAmica binding32. Before placing the fluid cell on top of the mica puck, we added an additional 1520 ml of 13 TAE/Mg21 buffer to the cavity between the fluid cell and the AFM cantilever chip to avoid bubbles. Fluorescence experiments. For catalytic circuitry experiments, we obtained fluorescence data using a QM-6/2005 steady state spectrofluorometer (Photon Technology International), equipped with a Turret 400TM four-position cuvette holder (Quantum Northwest) and 3.5-ml QS quartz cuvettes (Hellma). The temperature was set to 25 uC. We set the excitation and emission wavelengths to 520 nm (2-nm bandwith) and 540 nm (4-nm bandwidth), respectively. For the experiments in Fig. 3c, we prepared hairpin monomers, A, B, C and D, and initiator, I, separately as described above. We added 40 ml 1-mM A to 1.8 ml 13 TAE/Mg21 buffer and mixed it by rapid pipetting eight times using a 1-ml tip. We recorded the baseline signal for ,16 min. Then we added 40 ml of 1-mM B, C and D and the appropriate concentration of I (or 13 TAE/Mg21 buffer in the case of 03 I) to the cuvette (to reach the target concentrations described in Fig. 3c) and mixed by rapid pipetting eight times using a 1-ml tip. The control with 20-nM A alone was monitored continuously. The final volume was 2 ml for all experiments. We carried out the experiments with 10-mM inert 25-nt poly-T carrier strand21 in the individual hairpin and initiator stock solutions and ,1-mM inert 25-nt poly-T carrier strands in the final reaction solution. For autonomous locomotion experiments, we used the same spectrofluorometer as above with the temperature controller set to 21 uC. We used two 3.5-ml QS quartz cuvettes (Hellma) in each set of experiments. Excitation and emission wavelengths were set to 492 nm and 517 nm (for FAM), 527 nm and 551 nm (for JOE), and 558 nm and 578 nm (for TAMRA), respectively, with 4-nm bandwidths. The assembly of the walker system is described in Supplementary Information 6.4. We snap-cooled hairpin B in the reaction buffer (4 mM MgCl2, 15 mM KCl and 10 mM Tris-HCl, pH 5 8.0): heating at 95 uC for 90 s, rapid cooling at room temperature, sitting at room temperature for 30 min before use. The system was assembled using 4 nM track and 3.5 nM bipedal walker. We used a substochiometric amount of walker to ensure that no freefloating walker would bind to hairpin A on the track. For the same reason, we used substoichiometric monopedal walker (7 nM) in the diffusion experiments. The final concentration of hairpin B was 20 nM, which was equimolar with the five A hairpins on the track (5 3 4 nM 5 20 nM). The assembled track was first introduced to record the fluorescence baselines for FAM, JOE and TAMRA. We then introduced hairpin B and mixed 100 times by rapid pipetting to start walker locomotion.
31. Flamm, C., Fontana, W., Hofacker, I. L. & Schuster, P. RNA folding at elementary step resolution. RNA 6, 325338 (2000). 32. Hansma, H. G. & Laney, D. E. DNA binding to mica correlates with cationic radius: assay by atomic force microscopy. Biophys. J. 70, 19331939 (1996).
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SUPPLEMENTARY INFORMATION
Supplementary Information
Programming Biomolecular Self-Assembly Pathways

Peng Yin1,2 , Harry M.T. Choi1 , Colby R. Calvert1 & Niles A. Pierce1,3,
1 Department
of Bioengineering, 2 Department of Computer Science, 3 Department of Applied & Computational Mathematics California Institute of Technology, Pasadena, CA 91125, USA Email: niles@caltech.edu
Contents
S1 Summary gure S2 Reaction graph conventions S3 Catalytic geometry S3.1 System design for catalytic formation of a 3-arm junction S3.2 Execution of the reaction graphs . . . . . . . . . . . . . S3.3 Catalytic formation of a 4-arm junction . . . . . . . . . S3.4 AFM image analysis . . . . . . . . . . . . . . . . . . . S3.5 Design for the catalytic formation of a k-arm junction . . S4 Catalytic circuitry S4.1 Execution of the reaction graph . . . . . S4.2 Detailed secondary structure mechanism S4.3 Stepping gel . . . . . . . . . . . . . . . S4.4 System kinetics analysis . . . . . . . . S5 Nucleated dendritic growth S5.1 Execution of the reaction graph . . . . . S5.2 Detailed secondary structure mechanism S5.3 Quantitative amplication gel . . . . . . S5.4 AFM image analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 2 3 3 4 5 6 8 10 10 11 13 14 16 16 17 19 20 23 23 24 25 27 29 30 33 34 36 37 37 38 39 39 40 42 43 45 46
1
S6 Autonomous locomotion S6.1 Execution of the reaction graph . . . . . . . . . . . . S6.2 Secondary structure of the walker system . . . . . . S6.3 Detailed secondary structure mechanism . . . . . . . S6.4 Assembly of the walker system . . . . . . . . . . . . S6.5 Characterization of the fuel system . . . . . . . . . . S6.6 Raw data for the uorescence quenching experiments S6.7 Statistical analysis . . . . . . . . . . . . . . . . . . . S6.8 Comparison of walker time scales . . . . . . . . . . S6.9 Control for walker landing effects . . . . . . . . . .
S7 Discussion S7.1 Leakage and ligation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . S7.2 Molecular compiler . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . S8 DNA sequences S8.1 Catalytic 3-arm junction system . S8.2 Catalytic 4-arm junction system . S8.3 Autocatalytic system . . . . . . . S8.4 Nucleated dendritic growth system S8.5 Fuel for the walker system . . . . S8.6 Walker system . . . . . . . . . . .
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S1
Summary gure
Motif Molecular programs
Catalytic geometry
Molecular implementation
Molecular execution
10 nm
Catalytic circuitry
Exponential kinetics Signal 0
Nucleated dendritic growth
Time (hr)
Abstraction
30 nm
Autonomous locomotion
Signal
Sequential quenching
1 Time (hr)
Figure S1. Summary. Diverse biomolecular self-assembly (and disassembly) pathways are programmed using an abstraction of a versatile DNA
hairpin motif.
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S2
Reaction graph conventions
Using the denitions introduced in the main text, the reaction graph abstraction obeys the following conventions. Initial conditions The initial condition of the system is dened by the state of each port and the initial bonds between the ports. An initial bond between an output port and an input port implies that an assembly reaction has already occurred prior to the execution of the reaction graph (see, e.g., the bond between the output port of I and the input port of A in Fig. 5a). Static structural elements Static structural elements are depicted by gray line segments (e.g. the substrate of Fig. 5a) and are inert during execution of the reaction graph. These elements can be used to impose geometric constraints on the execution of the reaction graph (e.g. the rigid substrate and inextensible torso of the walker system, Sect. S6.1). Execution starting points Execution begins with any solid arrow (assembly reaction) connecting two accessible ports. In a system lacking two accessible ports connected by a solid arrow, execution cannot begin (e.g., the removal of node I prevents execution of all programs described in the present work). Assembly reaction An assembly reaction is depicted by a solid arrow that points from an input port to a complementary output port of a different node. An assembly reaction is executed when these two ports are simultaneously accessible. In the execution of an assembly reaction, a bond is formed between the two ports, they are ipped to their inaccessible states, and the internal logic of the node with the affected input port is applied to its output ports (e.g., for the present motif, the output ports are ipped to their accessible states). Multiple solid arrows entering the same input port depict parallel processes on separate copies of the nodal species (e.g., the input port of node A in Fig. 3a and the input ports of nodes A2-A5 and B2-B5 in Fig. 4a). Disassembly reaction A disassembly reaction is depicted by a dashed arrow that points from an input port to a complementary output port of a different node. Using nodal abstractions of the present hairpin motif, a disassembly arrow must complete a disassembly cycle. For a cycle involving k nodes: input port 1 blue output port 2 input port 3 blue output port 4 . . . blue output port 2k input port 1, where denotes an assembly reaction, denotes a disassembly reaction, and denotes the internal logical connection between two ports on the same node. For example, Fig. 1d contains a disassembly cycle for k = 2: input port of A blue output of A input port of B blue output port of B input port of A. Fig. 2a contains a disassembly cycle for k = 3: input port of A blue output port of A input port of B blue output port of B input port of C blue output port of C input port of A. In physical terms, this corresponds to requiring that the displacing strand and the strand to be displaced emanate as adjacent branches for a k-arm junction, allowing nucleation of the displacement branch migration (e.g., Figs 2b, S4b, and S7b). The special case of k = 2 corresponds to standard toehold-mediated strand displacement1 (e.g., Fig. 1b, where the whole of domain b of hairpin A serves as the toehold). A disassembly reaction is executed when the participating output port is accessible and the participating input port is inaccessible (using nodal abstractions of the present motif, the requirement that a disassembly arrow must complete a disassembly cycle implies that the participating output port can only become accessible after the participating input port becomes inaccessible). In the execution of a disassembly reaction (e.g., Fig. 1e), the existing bond from an (inaccessible) output port to an (inaccessible) input port is replaced by a new bond to the displacing (accessible) output port; the states of both output ports are ipped. Multiple dashed arrows entering the same input port depict parallel disassembly cycles involving separate copies of the nodal species (no such example is presented in this paper).
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S3
S3.1
Catalytic geometry
System design for catalytic formation of a 3-arm junction
a
Target dynamic function: Catalytic formation of a 3-arm DNA junction (1) Pathway specifiation
C
(2.1) Complementarity relationships
c
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I b* a
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c*
B c c*
a*
C a a*
b*
(2.2) Clamping/padding
I a* x* b* y*
A a x b y x* b* y* z* c*
B b y c z y* c* z* x* a*
C c z a x z* a* x* y* b*
(2.3) Dimensioning
e f
|a| = |b| = |c| = |x| = |y| = |z| = 6 nt
(3) Sequence design

DNA sequences A C G T
Figure S2. Procedure for designing the catalytic 3-arm junction system.
Here we describe the design procedure for the catalytic 3-arm junction system presented in Fig. 2. Step (1) Pathway specication. The desired dynamic behavior (Fig. S2a) is specied using a reaction graph (Fig. S2b).
Step (2) Translation into secondary structure motifs. The reaction graph can be translated directly into secondary structure motifs. Step (2.1) Basic complementarity relationships. In the reaction graph, two ports connected by an arrow are complementary to each other. These portal complementarity relationships specify the complementarity relationships between the motif
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domains modeled by the ports, and thus enable a direct translation of the reaction graph to the secondary structure motifs (Fig. S2c). For example, the assembly arrow connecting the brown output port of node I and the orange input port of node A (Fig. S2b) indicates these two ports are complementary, and hence the initiator and the orange domain of hairpin A in Fig. S2c are complementary. Similarly, the disassembly arrow connecting the blue output port of node C and the orange input port of node A (Fig. S2b) indicates these two ports are complementary, and hence the blue domain of hairpin C is complementary to the orange domain of hairpin A in Fig. S2c. Step (2.2) Clamping/padding. The basic implementation (Fig. S2c) is modied by adding clamping/padding segments (x, y, z, x*, y*, and z* in Fig. S2d). These segments serve two purposes. First, they serve as padding segments to modulate the lengths of a hairpins sticky-end, stem, and loop regions, permitting more exible dimensioning in Step (2.3). Second, the segments serve as clamps to decrease spurious leakage reactions in the absence of the initiators. Consider un-clamped hairpin A and hairpin B in Fig. S2c. When the left-end of the stem of hairpin A breathes, the 3 end of segment b* will be transiently exposed, revealing a partial toehold that is complementary to the toehold b of hairpin B. This transient toehold exposure would permit hairpin A and hairpin B to react spuriously and form AB (which would then react with C to form ABC). By contrast, the breathing of the left end of the clamped hairpin A stem in Fig. S2d exposes x* instead of b*. Thus, b* remains sequestered, discouraging spurious nucleation between A and B at b*. Step (2.3) Segment dimensioning. The purpose of segment dimensioning is to assign the length of each segment (number of nucleotides) such that under specied conditions, spurious reactions are suppressed and the desired reaction proceeds smoothly. The NUPACK server (www.nupack.org) is used for dimensioning. For the catalytic 3-arm junction system described here, the thermodynamic analysis of the interacting DNA strands suggests that assigning 6-nt to each segment (Fig. S2e) stabilizes critical structures in the reaction pathway in the context of a dilute solution of interacting nucleic acid strands.
Step (3) Sequence design. See Methods in main text.
S3.2
Execution of the reaction graphs

a
I (1) (4) C (3) A (2) B
(1)
A
(2)
B
A B
(3)
C
I C
A B
(4)
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A B
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I (1) (5) C (4) A (2) B
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I I A
(2)
I B
(3)
I C
(4)
I C D D
(5)
I C D
(3) D
Figure S3. Execution of reaction graphs for catalytic 3-arm/4-arm junction systems. a, Execution of the reaction graph of Fig. 2a. Reaction 1 (assembly): A bond is made between the accessible output port of I and the accessible input port of A and both ports are ipped to inaccessible states; the output port of A is ipped to the accessible state (based on the internal logic of node A). Reaction 2 (assembly): A bond is made between the newly accessible output port of A and the accessible input port of B and both ports are ipped to inaccessible states; the output port of B is ipped to the accessible state (based on the internal logic of node B). Reaction 3 (assembly): A bond is made between the newly accessible output port of B and the input port of C and both ports are ipped to inaccessible states; the output port of C is ipped to the accessible state (based on the internal logic of node C). Reaction 4 (disassembly): The bond between the inaccessible output port of I and the inaccessible input port of A is displaced by a bond between the newly accessible blue output port of C and the input port of A; the states of the two output ports are ipped. b, Execution of the reaction graph of Fig. 2e.
Fig. S3 depicts the step-by-step execution of the reaction graphs in Figs 2a and e. Note that the reaction graph in Fig. S3a contains a k = 3 disassembly cycle: input port of A blue output port of A input port of B blue output port of B input port of C blue output port of C input port of A; the reaction graph in Fig. S3b contains a k = 4 disassembly cycle: input port of A blue output port of A input port of B blue output port of B input port of C blue output port of C input port of D blue output port of D input port of A.
doi: 10.1038/nature06451
S3.3
Catalytic formation of a 4-arm junction

a
I (1) (4b) D
y* c* x* b* w* q*
(2)
B (3)
b
Initiator I a* w* b* x*
a w b x
Metastable monomers B A y* b x c y w* b* x* q* c* r* x* c* y*
C z* d* s* c y d z y* d* z* w* a*
D d z a w z* a* w* t*
x* b*
(4a)
C
q* q* q* y* c* x* b* w* y c x b A.B.C.D y c x b z* d* y* c* x* s* y* d* z* a* w* z d y c
y* c* x* b* w*
y* c* x* b* w*
y c x b
y c x b
I.A I a* w* b* x* (1)
I.A.B
I.A.B.C
I.A.B.C
y* c* x* b* w*
Cataytic formation of a 4-arm junction
q*
a w b x a* w* b* x* (2)
a w b x a* w* b* x*
z* d* y* c* x*
r* (3)
a w b x a* w* b* x*
z* d* y* c* x* y* d* z* a* w* z d y c
r* (4a)
a w b x a* w* b*x*
z* d* y* c* x* y* d* z* a* w* z d y c
r* (4b) t*
a w b x z* a* w* b* x* d z a w
r*
a* w* b* x* d z a w
I a* w* b* x*
s*
z*
s*
t*
Extend arms for AFM imaging AFM imaging A.B.C.D.Ae.Be.Ce.De y* c* x* b* w* q* q Ae q Be r Ce y De z t s
c
I A+
+B I+A +B +C I+A +B +C +D 0.5 I+A +B 0.2 +C 5 +D I+A +B 0.1 +C I+A +D +B A+ +C B+ +D C+ D A+ B+ C+ D.a nn ea led
Step 4 Step 3
y c x b w
Step 2 Step 1
I+A
x x* x r* r
10
Figure S4. Catalytic formation of a 4-arm DNA junction. a, Reaction graph. Note that since the green output ports do not serve as initiators for any downstream reaction, they are omitted here for simplicity. See Sect. S3.2 for step-by-step execution of the graph. b, Secondary structure schematic of the reaction. The lengths of segments q, q*, r, r*, s, s*, t, and t* are 18 nt; the lengths of the other segments are 6 nt. Hairpins A, B, C, and D are metastable in the absence of the initiator I. The initiator I catalyzes monomers A, B, C, and D to form a 4-arm DNA junction, as follows: (1) segment a* of I nucleates at the toehold a of hairpin A and initiates a strand displacement that results in the opening of hairpin A; (2) newly exposed b* of A nucleates at toehold b of B and results in the opening of B; (3) newly exposed c* of B nucleates at toehold c of C and results in the opening of C; (4a) newly exposed d* of C nucleates at d of hairpin D and results in the opening of D; (4b) D displaces I from A. c, Agarose gel electrophoresis demonstrates the catalytic formation of the 4-arm junction. Lanes 1-5: A gel shifting assay validates each reaction step depicted in panel (b). Lanes 5-9: Effects of different concentrations of I (1, 0.5, 0.25, 0.1, and 0) on the formation of ABCD. 600 nM reactants were incubated at room temperature for 2 hours. Lane 10: ABCD annealed over 2.5 hours (600 nM hairpin species heated at 95 C for 5 minutes and cooled to room temperature over 2.5 hrs). The 2% agarose gel was prepared in 1 LB buffer (Faster Better Media, LLC) with 0.5 g/ml ethidium bromide. The gels were run at 150 V for 30 min at room temperature and then visualized using UV transillumination. The hairpins used for these reactions did not contain the 3 tails (q*, r*, s*, and t*). d, AFM images of two 4-arm junctions. To assist in AFM imaging of the 4-arm junction, four strands (Ae, Be, Ce, and De) were incubated with the catalytically formed 4-arm junction ABCD. Note that the duplex portion of the arms of the nal structure ABCDAeBeCeDe are twice as long as the duplex portion of the arms of ABCD. Two AFM images of ABCDAeBeCeDe are presented. Scale bar, 10 nm. See Sect. S3.4 for length measurements.
Fig. S4a and b depict the reaction graph and reaction schematic for the catalytic formation of a 4-arm junction. In the absence of initiator I, hairpins A, B, C, and D are kinetically impeded from forming the 4-arm junction that is predicted to dominate at equilibrium. Introduction of I into the system (Fig. S4b, bottom) activates a cascade of assembly steps with A, B, C, and D followed by a disassembly step in which D displaces I from the complex, freeing I to catalyze the self-assembly of additional branched junctions. Native agarose gel electrophoresis (Fig. S4c) conrms that the hairpins assemble slowly in the absence of the initiator (Lane 9) and that assembly is dramatically accelerated by the addition of initiator (Lane 5). Disassembly of the initiator enables catalytic turnover as indicated by the nearly complete consumption of hairpins even at sub-stiochiometric initiator concentrations (Lanes 6-8). As in the 3-arm junction case, only minimal assembly is achieved by annealing the hairpin mixture (Lane 10). AFM imaging of the catalyzed self-assembly product (augmented with strands that extend the duplex portion of each arm as described in the caption) reveals the expected 4-arm junction morphology (Fig. S4d).
s*
y* d* z* a*w*
d z a w
A.B.C.D I.A.B.C I.A.B I.A A
Leakage t t* z
a w b x
z* d* y* c* z d y c
z* a* w* b* x*
doi: 10.1038/nature06451
S3.4
AFM image analysis
a
10nm 10nm
7.7 nm
~7.8 nm
8.1 nm
7.7 nm
7.1 nm
7.7 nm
7.7 nm
b
10nm 10nm
6.6 nm
7.1 nm
7.0 nm
12.5 nm
14.3 nm
8.2 nm
~7.8 nm ~7.8 nm
7.6 nm
14.5 nm
14.2 nm
7.1 nm
7.5 nm
7.0 nm
Figure S5. AFM image analysis of 3-arm/4-arm junctions. AFM measurements of the 3-arm (panel a) and 4-arm (panel b) junctions described
in Fig. 2 and Fig. S4. The small images are screenshots of the measurement section les. The distance between the two arrows is listed above the image.
Using a B-DNA model where one helical turn contains 10.5 base pairs and measures 3.4 nm, we calculate the expected arm length for the 3-arm junction as follows: (24 / 10.5) 3.4 nm = 7.8 nm. Similarly, the arm length for the 4-arm junction is calculated to be 7.8 + 7.8 = 15.6 nm. The measured lengths of the arms are roughly consistent with the calculated lengths (Fig. S5). Fig. S6 shows AFM images with a larger eld of view for 3-arm (panel a) and 4-arm (panel b) junctions.
doi: 10.1038/nature06451
100 nm
100 nm
Figure S6. Large-eld-of-view AFM images of the 3-arm (a) and 4-arm (b) junction systems.
doi: 10.1038/nature06451
S3.5
Design for the catalytic formation of a k-arm junction
b
H2 H3
Initiator
a* 1 x * 1 a* 2 x * 2
Catalytic formation of a k-arm junction

I
a* 1 x * 1 a* 2 x * 2
I
a
I H1
H1
x*
4
a1
x1
a2
x2
x*3
Hk-2
a k-2 x k-2 a k-1 x k-1 x * k-2 a* k-1 x * k-1

Hk-1
x*k
H2
a2
Hk Hk-1
x2
a3
x3
x*4
a k-1 x k-1 a k x * k-1 a* k

Hk
xk x*k
x*1
a1
x1 a2 x2
x*
...
x * 1 a* 2 x * 2
a* 3
a* k
H1.H2....Hk
a*
3 a *3
x* a2 x2
Metastable monomers
x * k a* 1 x * 1 a* 2 x * 2
x1
x* 1 a* 2 x* 2
...
a3 x3 x* 3
a* 3
x k-1 a k-1 x k-2 a k-2
...
x* k
xk
a1
x*
x * 2 a* 3 x * 3
a* k
a* 4
a* 1 x*2
ak
x* k
k a *1
xk
x k-
-1
a*
......
H3 Hk-3
a* k
x*
ak
xk
a1
x1
ak
a k-
-1
x* k
Figure S7. Catalytic formation of a k-arm junction. a, Reaction graph. b, Reaction schematics. Hairpins H1 , H2 , . . . , Hk are metastable in the
absence of the initiator I. The initiator I catalyzes monomers H1 , H2 , . . . , Hk to form a k-arm DNA junction.
The catalytic system described in Fig. 2 and Fig. S4 can, in principle, be generalized to a system capable of the catalytic formation of a k-arm junction. Fig. S7 describes the reaction graph and the secondary structure schematic for the catalytic formation of a k-arm junction. Fig. S8 gives an example when k = 6.
x*
x * k a* 1 x * 1
k-1
-2
a* 2
doi: 10.1038/nature06451
b
H2 H3
Initiator
a* 1 x * 1 a* 2 x * 2
Catalytic formation of 6-arm junction a* 1 x * 1 a* 2 x * 2

I
Metastable monomers
H1
a
I
(3)
(2) (1)
H1
(4)
H4
a1
x1
a2
x2
x*3
H4
a4
x4
a5
x5
x*6
(1)
x* 1
x * 1 a* 2 x * 2
H2
a* 3 x*4
x * 4 a* 5 x * 5
H5
a* 6
x*1
a* 2
(7)
H6
(5) (6) a2
H5
x* 2
x2
a3
x3
a5
x5
a6
x6
a* 3 x* 3
I.H1
x * 2 a* 3 x * 3
H3
a* 4 x*5
x * 5 a* 6 x * 6
H6
a* 1
a1
x1 a2 x2
a3
x3
a4
x4
a6
x6
a1
x1
x*2
a* 1 x * 1 a* 2 x * 2
x * 3 a* 4 x * 4
a* 5
x * 6 a* 1 x * 1
(2) a* 2
x*
x*
3 a *3
3 a *3
a3
a3
4 x *
4 x *
x3
x3
a*
a*
a4
a4
x*
I.H1.H2.H3
x*
x4
x4
a1
x1 a2 x2
x * 5 a* 5 x * 4 a* 4 x * 3 x5 a5
a* 6
a1
x1 a2 x2
x * 5 a* 5 x * 4 a* 4 x * 3
a1
x1 a2 x2
a* 1 x * 1 a* 2 x * 2
x4 a4
a* 1 x * 1 a* 2 x * 2
a* 1 x * 1 a* 2 x * 2
(5)
a* 1 x * 1 a* 2 x * 2
I
x*
I.H1.H2.H3.H4
I.H1.H2
(4)
(3)
a*
4 x * 4 x *3
3 x *2
3 a *3
a*
a3
a* a* x* x4
4 x *
4 x *3
a3
x3
x3
a*
a4
a*
a4
x*
x4
x*
a1
x1 a2 x2
x * 5 a* 5 x * 4 a* 4 x * 3 x5 a5
a* 6
x4
(6)
a1
x1 a2 x2
x * 5 a* 5 x * 4 a* 4 x * 3 x5 a5
a* 6
(7)
a1
x1 a2 x2
x * 5 a* 5 x * 4 a* 4 x * 3 x5 a5
a* 6
a* 1 x * 1 a* 2 x * 2
1
x4 a4
a* 1 x * 1 a* 2 x * 2
x1 a* 2 a6
1 x *
x4 a4
a4
I.H1.H2.H3.H4.H5
I.H1.H2.H3.H4.H5.H6
H1.H2.H3.H4.H5.H6
x3
a3
3 x *2
3 a *3
x* a2 x2 x* 2 a3 a* 3 x3 x* 3 a2 x2 x* 2 a3 a* 3 x3 x* 3
x* 1
x* 1
x1
1 x *
1 x *
a1
a1
a*
a*
x6
x6
a*
x*
x*
a6
x*
a*
a*
a*
x*
Figure S8. Catalytic formation of a 6-arm junction. a, Reaction graph. b, Step-by-step reaction schematic. Hairpins H1 , H2 , H3 , H4 , H5 , and
H6 are metastable in the absence of the initiator I. The initiator I catalyzes monomers H1 , H2 , H3 , H4 , H5 , and H6 to form a 6-arm DNA junction as follows. Step 1: segment a of I nucleates at the toehold a1 of hairpin H1 and initiates a strand displacement that results in the opening of 1 hairpin H1 . Step 2: the newly exposed a of H1 nucleates at the toehold a2 of hairpin H2 and opens hairpin H2 . Step 3: the newly exposed a 2 3 of H2 nucleates at the toehold a3 of hairpin H3 and opens hairpin H3 . Step 4: the newly exposed a of H3 nucleates at the toehold a4 of hairpin 4 H4 and opens hairpin H4 . Step 5: the newly exposed a of H4 nucleates at the toehold a5 of hairpin H5 and opens hairpin H5 . Step 6: the newly 5 exposed a of H5 nucleates at the toehold a6 of hairpin H6 and opens hairpin H6 . Step 7: H6 displaces I from H1 . 6
x*
x*
x* 1 a* 2 x* 2 x* 1 a* 2 x* 2
a2
a2 x2 a3 x3 x2 a3 x3 x* 3 x6 x* 3 a6 a2 a* 3 a* 3
a* 2
a* 2 x* 2
x2 a3 x3 x* 3
a* 3
x* x* x5
6 x* 5 a* 5 x* 4 x* 1 x* 1 a* 2 x* 2 a* 3 x* 3 a2 x2 a3 x3 a* 2 x*
x * 6 a* 1 x * 1 a* 2 x * 2
x6
x4 a4
x*
6 x* 6 a* 1 x* 1 x* 5 a* 5 x* 4 a5
x* 2
x6 a6
a6
x* 5 a* 5 x* 4
x* 5
x5 a5
x5 a5
a* 5 x* 4
10
doi: 10.1038/nature06451
S4
S4.1
Catalytic circuitry
Execution of the reaction graph
I I A A
I (1) A (5) D (2a) (6a) (4b) (4a) (2b) (6b) B (3) C
(1)
(2a)
B
(2b)
A
B
A
B
(3)
C C D
(4a)
(4b)
(5)
D C A D C
(6a)
B
(6b)
Figure S9. Execution of the reaction graph for the autocatalytic system of Fig. 3.
Fig. S9 describes the step-by-step execution of the reaction in Fig. 3a. The reaction starts at solid arrow (1) that connects the accessible output port of I and the accessible input port of A. Note that by convention, the two arrows entering the same input port of A depict parallel processes on separate copies of the nodal species. Reaction 1 (assembly): A bond is made between the accessible output port of I and the accessible input port of A and both ports are ipped to inaccessible states; the output port of A is ipped to the accessible state (based on the internal logic of node A). Reaction 2a (assembly): A bond is made between the newly accessible output port of A and the accessible input port of B and both ports are ipped to inaccessible states; the two output ports of B are ipped to accessible states (based on the internal logic of node B). Reaction 2b (disassembly): The bond between the inaccessible output port of I and the inaccessible input port of A is displaced by a bond between the newly accessible blue output port of B and the input port of A; the states of the two output ports are ipped. Reaction 3 (assembly): A bond is made between the newly accessible green output port of B and the accessible input port of C and both ports are ipped to inaccessible states; the output port of C is ipped to the accessible state (based on the internal logic of node C). Reaction 4a (assembly): A bond is made between the newly accessible output port of C and the accessible input port of D and both ports are ipped to inaccessible states; the output ports of D are ipped to accessible states (based on the internal logic of node D). Reaction 4b (disassembly): The bond between the inaccessible green output port of B and the inaccessible input port of C is displaced by a bond between the newly accessible blue output port of D and the input port of C; the states of the two output ports are ipped. Reaction 5 (assembly): A bond is made between the newly accessible green output port of D and the accessible input port of A and both ports are ipped to inaccessible states; the output port of A is ipped to the accessible state (based on the internal logic of node A). Reaction 6a (assembly): A bond is made between the newly accessible output port of A and the accessible input port of B and both ports are ipped to inaccessible states; the output ports of B are ipped to accessible states (based on the internal logic of node B). Reaction 6b (disassembly): The bond between the inaccessible green output port of D and the inaccessible input port of A is displaced by a bond between the newly accessible blue output port of B and the input port of A; the states of the two output ports are ipped.
10
doi: 10.1038/nature06451
S4.2
Detailed secondary structure mechanism

Metastable monomers Initiator
I A
a x v b y x* v* b* y*
x*
a* c*
b y u c a x
y* b*
c u y d v
u* y* d* v*
u*
c* a*
d v x a c u
v* d*
a* x* v* b* y*
u*
y* u* c* a* x* v* d* v*
x*
v* x* a* c* u* y* b* y*
Self-replication
I a* x* v* b* y*
x*
a* c*
I.A a
y x* a* c* u* y* b* v* x*
u*
(1)
a* x* v* b* y*
(2)
b y u c a x y* b*
B
A.B
x* a* c* u* y* b* v* x* a c u
v* d* y* u* c* a* x* v* b* y* x B
y* u* c* a* x* v* d* v* c u y d v u* c* a*
b y u c a x
y* b*
v* d* y* u*
C
y* u* c* a* x* v* d* v*
C.D.A u* y* d* v* x* a* c* u* v d v x a c d y u
(6)
a x v b
(3)
c* a*
u* y* d* v*
x*
x*
x* a*
v* d* y* u* c* a* x* v* b* y*
u*
x* a* c* u* y* b* v* x* a c u
c*
u* y*
v* d* y* u* c* a* x* v* b* y* x
A.B.C
v* b*
x*
a* c*
(5)
(4)
d v x a c u
d y u
v*
x*
d*
c
u*
u* y* d* v* x* a* c* u* v C.D d v x a c
v* x* a* c* u* y* b* y*
v* d* y* u* c* a* x* v* b* y*
Figure S10. Detailed reaction schematic for the autocatalytic system of Fig. 3. The length of each segment is 6 nt. Green star, uorophore;
black dot, quencher.
Fig. S10 describes the detailed reaction ow of the autocatalytic system described in Fig. 3. Fig. S11 describes additional intermediate steps. Steps 1-2 are the initiation stage; steps 3-6 are the exponential amplication stage. Step 1: the toehold a* of I nucleates at the toehold a of A, resulting in the opening of the hairpin and the formation of the product IA. Step 2: IA, with b* newly exposed, opens hairpin B (step 2a); B subsequently displaces I from A (step 2b), producing AB and bringing the system to the exponential amplication stage. The single-stranded tail (v*-d*-y*-u*-c*) of AB next catalyzes C and D to form CD (in steps 3 and 4). Step 3: AB, with c* newly exposed, opens hairpin C. Step 4: ABC, with d* newly exposed, opens hairpin D (step 4a); D subsequently displaces C from B, separating AB and CD (step 4b). The single-stranded tail (a*-x*-v*-b*-y*) of CD is identical to I and next catalyzes A and B to form AB (in steps 5 and 6). Step 5: CD, with a* newly exposed, opens hairpin A. Step 6: CDA, with b* newly exposed, opens B (step 6a); B subsequently displaces A from D, separating CD and AB (step 6b).
12
11
doi: 10.1038/nature06451
Step (1)
a x A x* v* b* y* v b y
x*
a* c* (1) I.A a x v b
y x* a* c* u* y* b* v* x*
+
I
u*
a* x* v* b* y*
a* x* v* b* y* a a x v b x v b
Step (2)
I.A
y x* a* c* u* y* b* v* x*
c u
y x* a* c* u* y* b* v* x*
I.A.B
b* y*
a* x* v* b* y* x a
b a A.B (2b) v* d* y* u* c* a* x* v* b* y* x a c u x v b
a* x* v* b* y*
x* a* c* u* y* b* v* x*
b B
y u c a x
b* y* u* c* a* x* v* d* v*
c*
a*
y*
x*
v*
(2a)
+
I a* x* v* b* y*
Step (3)
A.B a x v b
x* a* c* u* y* b* v* x* a u* c u c* a* x*
v*
d*
y*
u*
y* u* c* a* x* v* d*
v* d* y* u* c* a* x* v* b* y* x
b (3) A.B.C
+
c u y d v C
u*
u* y* d* v*
x* a* c* u* y* b* v* x* a c u
v* d* y* u* c* a* x* v* b* y* x
Step (4)
y* u* v* d* x* c* a* u*
v
d y u A.B.C u* y* d* v* x* a* c* u* v A.B.C.D d v x a c d y u
x* a* c* u* y* b v* x* a c u
u v* d* y* u* c* a* x* v* b* y* x
v*
x* a* c* u* y* b* v* x* a v* d* c u
d* y*
v* d* y* u* c* a* x* v* b* y* x
(4a)
u*
+
d v x a c u D
c*
a* x* v*
v* x* a* c* u* y* b* y* a x v b
A.B (4b) C.D d v x
v* d* y* u* c* a* x* v* b* y* x
b*
y*
x* a* c* u* y* b* v* x* a u c u
u* y* d* v* x* a* c* u* v a c
v* d* y* u* c* a* x* v* b* y*
Step (5)
u* y* d* v* x* a* c* u* v C.D d v x a c d y u
c
C.D.A
u* y* d* v* x* a* c* u* v d v x a c
x* a*
v* d* y* u* c* a* x* v* b* y*
v* d* y* u* c* a* x* v* b* y*
c*
+
a x A x* v* b* y* v b y
(5)
u*
x*
y*
a* c*
v* b*
x*
u*
Step (6)
u* y* d* v* x* a* c* u* v d C.D.A b B v x a c d y u
u* y* d* v* x* a* c* u* v
x* a* c* u* y* b* v* x* x a c u
+
y u c a x y* b* y* u* c* a* x* v* d* v*
A.B (6b)
v* d* y* u* c* a* x* v* b* y* x
v*
x* a* c* u* y* b* v* x* a u c u
d*
y*
u*
c*
a*
x*
(6a)
v*
b*
v* d* y* u* c* a* x* v* b* y*
+
u* y* d* v* x* a* c* u* v C.D d v x a c d y
v* d* y* u* c* a* x* v* b* y*
Figure S11. Step-by-step reaction schematic for the autocatalytic system of Fig. 3.
y*
C.D.A.B
x* a* c*
v* d* y* u* c* a* x* v* b* y*
u* y* v* b*
x*
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S4.3
Stepping gel
Self-replication A
A.I
(1)
I A.B Initiation
(2)
B
(6)
Exponential amplification
(3)
A .B .C
C.D.A
(5)
A C.D
(4)
D
)+C (AB C) (AB C)+ D (CD )
A.B.C A.B.C A.B A.B A .I A A.I
C.D.A C.D.A
A.B, C.D C.D
1 2 3 4 5 6 7 8 9 10 11 12 13 14 Figure S12. Stepping gel for the autocatalytic system. a, Reaction schematic. b, Native polyacrylamide gel electrophoresis demonstrates the
step-by-step reaction depicted in Fig. 3b. The symbol () indicates annealing; + indicates 15 minute reaction at room temperature. The hairpins used for these reactions were synthesized and puried by IDT DNA and used without further purication. The annealed samples were annealed at 2 M reactant concentrations: heating at 95 C for 5 minutes followed by cooling to room temperature over approximately 2.5 hours. The room temperature reactions were conducted with each reactant species at 1 M concentration. Consider the sample, (AI) + B, in Lane 5. The sample was prepared by rst annealing a mix containing 2 M A and 2 M I to produce (AI). Then 2 L of (AI), at 2 M concentration, was mixed with 2 L of B at 2 M concentration and allowed to react at room temperature for 15 minutes. Lanes 1 and 14 are 20-1000 bp DNA ladders (Bio-Rad). The 5% native polyacrylamide gel was prepared in 1 TAE/Mg++ buffer (20 mM Tris, pH = 7.6, 2 mM EDTA, 12.5 mM Mg++ ). The samples were loaded with 10% glycerol. The gel was run at 100 V for 90 minutes at room temperature, post-stained with 0.5 g/mL ethidium bromide, and visualized by UV transillumination. The blue line delineates the boundary between two gels.
The autocatalytic system was validated on a step-by-step basis using native polyacrylamide gel electrophoresis (PAGE) (Fig. S12): Step 1. Hairpin A reacts with initiator I and produces a band that corresponds to product AI (Lane 3), which migrates at about the same speed as the annealed product AI (Lane 4), as expected. Step 2. Annealed sample AI reacts with hairpin B and produces a band that corresponds to product AB (Lane 5), which migrates at about the same speed as the annealed product AB (Lane 6), as expected. Step 3. Annealed sample AB reacts with hairpin C and produces a band that corresponds to product ABC (Lane 7), which migrates at about the same speed as the annealed product ABC (Lane 8), as expected. Step 4. Annealed sample ABC reacts with hairpin D and produces a band that corresponds to product AB and CD (Lane 9), which migrates at about the same speed as the annealed product AB (Lane 6) and the annealed product CD (Lane 10), as expected. Step 5. Annealed sample CD reacts with hairpin A and produces a band that corresponds to product CDA (Lane 11), which migrates at about the same speed as the annealed product CDA (Lane 12), as expected.
13
(CD )+A (CD A)
(AI)
(AI)
(AB
(AB
C.D, A.B
(CD A)+ B
Step 1
Step 2
+B
Step 3
Step 4
Step 5
Step 6
A+I
doi: 10.1038/nature06451
Step 6. Annealed sample CDA reacts with hairpin B and produces a band that corresponds to product CD and AB (Lane 13), which migrates at about the same speed as the annealed product CD (Lane 10) and the annealed product AB (Lane 6), as expected.
S4.4
System kinetics analysis
Analytical modeling The autocatalytic system is modeled using the following reactions:
1 I + A IA
IA + B AB + C ABC + D CD + A CDA + B
2 I + AB 3 ABC 4 AB + CD 5 CDA 6 CD + AB.
k k k k k
To make the system tractable for analytical treatment, we make the following simplifying assumptions: Assumption 1. The forward reaction rates are all the same: ki = k, for i = 1, . . . , 6. This is based on the fact that all the reactions are strand-displacement reactions mediated by 6-nt toe-holds. Under the experimental conditions, the rate limiting step of the toe-hold mediated reactions is the nucleation step,2 the rate of which is determined primarily by the toe-hold length. Assumption 2. The reactions are irreversible. This approximation is justied by the fact that at 25 C, the equilibrium Q constants for these reactions (e.g., K1 QIIA for the rst equation here, Q denotes the partition function for a given QA complex species) are all calculated to be greater than 109 using multi-stranded partition function analysis3 with NUPACK (www.nupack.org). Assumption 3. CD and I are treated as identical species at the level of mass action kinetics modeled here. This is based on the fact that the active catalytic segment of CD is identical in sequence to I (see Fig. S10). Under the above assumptions, the model may be simplied to AB + C ABC + D CD + A CDA + B
k k k k
ABC AB + CD CDA CD + AB.
The kinetics of the system can be modeled by the following equations: d[ABC] dt d[CDA] dt d[AB] dt d[CD] dt = k[AB][C] k[ABC][D] = k[CD][A] k[CDA][B] = k[ABC][D] + k[CDA][B] k[AB][C] = k[ABC][D] + k[CDA][B] k[CD][A].
We next analyze the initial reaction stage of the system when the hairpin reactant depletion is small. Assumption 4. Initial stage assumption. [A] = [A]0 = [B] = [B]0 = [C] = [C]0 = [D] = [D]0 = c0 .
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Let = k[A]0 = k[B]0 = k[C]0 = k[D]0 = kc0 . Solving the above equations under the initial condition, [AB]0 = [CDA]0 = [ABC]0 = 0 and [CD]0 = [I]0 , we nd [ABC] = [CDA] = [AB] = [CD] =
1 [I]0 et ( 2e 2t (1 e2 2t ) 4t) 8 1 [I]0 et ( 2e 2t (1 e2 2t ) + 4t) 8 1 [I]0 et (e 2t (1 + e2 2t ) 2) 4 1 [I]0 et (e 2t (1 + e2 2t ) + 2). 4
Denoting by f the measurable uorescent species, we have f = [ABC] + [CDA] + [AB] 1 1 [I]0 et ( 2e 2t (1 e2 2t )) + [I]0 et (e 2t (1 + e2 2t ) 2). = 4 4
For the 6-nt toe-hold mediated strand displacement reactions analyzed here, the reaction constant k is estimated4to be 1 1 0.1 > e 2t , so the 105 M1 s1 . For c0 = 20 nM, we have = 2 103 s1 . When t 815 s, we have e 2t > 10 above equation may be simplied to 2 1 [I]0 et e 2t + [I]0 et e 2t f 4 4 1+ 2 = [I]0 e( 21)t . 4 This implies that the time, t, required to achieve a given uorescence level, f , is log-linear in [I]0 . Data analysis The analytical prediction that there is a regime in which t is log-linear in [I]0 is experimentally veried by the linear t of the 10% completion time against the logarithm of the relative concentration of I, 0.003 [I] 0.05 (Fig. 3c, inset; Fig. S13, blue circles). High concentration end points ([I] 0.1) are excluded based on theoretical analysis (t 815 s).1 The low concentration end points ([I] 0.001) are excluded due to signal poisoning by leakage. A system with no leakage would be expected to maintain the linear regime to arbitrarily low initiator concentrations. We can model our leaky system by augmenting the initiator concentration [I] by an additional leakage term to obtain an effective concentration, [I]e = [I] + [I]leak . With [I]leak = 0.005, a linear regime is observed between the 10% completion time and the logarithm of [I]e for [I] 0.05 (Fig. S13, red diamonds).
1.5
1.0
Time (hr)
0.5
-3
-2 log10 [I]
-1
Figure S13. Linear t of the 10% completion time against the logarithm of the relative concentration of I. Blue circles represent the initiator
concentration, [I] (Note that [I] = 0 is not shown); red diamonds represent the effective concentration of the initiator, [I]e = [I] + [I]leak with [I]leak = 0.005.
10% completion time for [I] = 0.05 is 610 < 815 s. This data point is nonetheless included: e 2t = 5.61 for t = 610 s and the approximation 2 induces an error of 1+ 2t = 14.8%. By comparison, all the excluded high concentration end points have 10% completion time t < 140 s.
1 The 2e
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S5
Nucleated dendritic growth
S5.1

I
(1)
I A1
(2)
I A1
(3)
I A1
I (1) A1 A2 A3 A4 A5 (2) (3) (4) (5)
A1
A2 B2 A2
A3 B3 B2 A3 A2 B3
B2 A3 B3
B2 B3 B4 B5
(4)
I A1
(5)
I A1
A4 B4
A5 B5
A2 A3 A4 B4 A4 B3 B4 A4 A3
B2 B3 B4 A4 B4 A4 A3
A2 B3 B4 A4 B4 A4 A3
B2 B3 B4 A4 B4
A5 B5 A5 B5 A5 B5 A5 B5 A5 B5 A5 B5 A5 B5 A5 B5 Figure S14. Execution of the reaction graph for the nucleated dendritic growth system.
Fig. S14 depicts the execution of the reaction graph of Fig. 4a. We omit a detailed description of the reaction graph execution, but note: (1) the multiple arrows entering the same input port depict parallel processes on separate copies of the nodal species, (2) the parallel processes are not synchronized and hence it is possible, for example, that after A1 assembles with A2, the assembly of A2 with A3 occurs before the assembly of A1 with B2.
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S5.2

Metastable monomers
A1 a x c b x y x* d* x* c* b* x* d* e* x* e* f* g* x* x* e* d* x* g* i* x* x* h* B3 i* g* c x d e x y x* e x f g x y x* A2 b x e d x y x* f* x* g* f* x* i* h* A3 d x g f x y x* h* A4 f j* k* x* x i h x y x* j* x* i* h* x* k* x* k* j* x* m* A5 h x k j x y x* l*
Initiator
I a* x* c* b* x*
x* f* f* B2 g* x* d* e* x*
h* x* f* g* x* i*
x* j* j* B4 k* x* h* i* x* k* g x h i x y x*
B5
l* x* j* k* x* m* k x y x*
i x j
Nucleated growth of a binary tree (part I)

x*
b*
I a* x* c* b* x* (1)
x* c*
a*
x a
c b A1 x y x* d* e* x* b* c* x* d* e* x*
x*
(2)
c* b*
b
x*
c
a*
x a
x y x* d* e* x* b* c* x* d* e* x*
y x d e x b c x d e x y x* x* f* g* g* f* x* x* d* A2 B2 e* G2 e* d* x* x* f* g* g* f* x* x*
A1
G1
a x c b A1 x y x* d* e* x* b* c* x* d* e* x*
(3) x* i* h* x* g* f* x* i* h* x* y x f g x d e x f g B3 x x* h* i* x* f* g* x* h* i* x* y A3 y x d e x b c x d e x y x* x* f* g* g* f* x* x* d* A2 B2 e* e* d* x* x* f* g* g* f* G3 x* x*
b* x*
c*
x*
a*
y x* i* h* x* g* f* x* i* h* x* B3 g f e d g f x x x x
(4)
A3 y x* h* i* x* f* g* x* h* i* x*
Figure S15. Reaction schematic of the nucleated dendritic growth system (part I). Step-by-step reaction schematic of the nucleated dendritic growth system, as described in Fig. 4. The lengths of segments x, x*, and y are 2 nt; the lengths of the other segments are 7 nt. The gure continues in Fig. S16.
Fig. S15 and Fig. S16 present the detailed reaction schematic of the nucleated dendritic growth system described in Fig. 4. In the absence of the initiator I, hairpin monomers co-exist metastably. The initiator I triggers the system to self-assemble into a binary tree of a prescribed size. Step 1: the toehold a* of the initiator I nucleates at the toehold a of hairpin A1, resulting in the opening of A1 and the formation of the rst generation dendrimer, G1. Step 2: A1, with b* and c* newly exposed, opens hairpins A2 and B2, producing the second generation dendrimer, G2. Note that now A2 and B2 reveal single-stranded tails of identical sequences. Step 3: A2 and B2, with d* and e* newly exposed, open hairpins A3 and B3, producing G3. Step 4: each copy of A3 and B3, with its newly exposed f* and g*, opens hairpins A4 and B4, producing G4. Step 5: each copy of A4 and B4, with its newly exposed h* and i*, opens hairpins A5 and B5, producing G5.
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Nucleated growth of a binary tree (part II)

x* j* k* x* h* B4 A4 i* x* j* k* x* y x i h x g f x i h x x* k* j* x* i* h* x* k* j* x* x* j* k* x* h* i* B4 x* j* k* x* x i h x g f x* k* j* x* i* A4 h* x* k* j* x* h x
x*
c* b*
I a* x*
x y x* d* e* x* b* c* x* d* e* x*
A1
y
y x* i* h* x* g* f* x* i* h* x* B3 g f e d g f x x x x
(4)
x* i* h* x* g* f* x* i* h* x* y x A3 f g x d e x f g B3 x x* h* i* x* f* g* x* h* i* x* y y y x h i x f g x h i x x* x* k* j* j* k* x* x* i* h* A4 B4 h* i* x* x* j* k* k* j* x* x*
y x d e x b c x d e x y x* x* f* g* g* f* x* x* d* B2 e* A2 e* d* x* x* f* g* g* f* x* x*
(5)
A3 y x* h* i* x* f* g* x* h* i* x* y g x h i x x* k* j* x* i* B4 h* x* k* j* x*
y x h i x f x* j* k* x* h* i* A4 x* j* k* x* G4
x*
x* k*
k*
j*
j* x*
x*
x* k* j* x* m* l* x* y x A5 j k x h i x j k B5 x x* j* k* x* l* m* x* y
B5 x* j* x* k* x* k* j* x* j* h* k* I B4 x* * i* x* B5 a A5 m* x* x* l* c* l* b* a j* A4 x m* x* x* c k* x* b A1 x x* y y x* d* e* x* b* c* x* d* e* x* y x i h x g f x i h x
m *l *
A5
x*
B4 x i
x i h x g f
x* k* j* x* i* A4 h* x* k* j* x* h x
y x* m* l* x* k* j* x* x B5 k j x i h x k j A5 x y x* l* m* x* j* k* x*
* m l* x*
x*
yx
x*
x*
j*
k*
k*
j*
x*
x*
h*
i*
h*
i* * x
k*
j*
j*
k* x*
x*
e d g f x x
x* k* j* x* m* l* x* y x A5 j k x h i x j k B5 x x* j* k* x* l* m* x*
Figure S16. Reaction schematic of the nucleated dendritic growth system (part II). Step-by-step reaction schematic of the nucleated dendritic
growth system, as described in Fig. 4. The gure continues from Fig. S15.
x*
j*
k*
G5
x*
l*
x h i x f g x h i x x* j* k* B4 x* h* A4 i* x* j* k* x* A5
k* j* x* i*
y x* l* m* x* A5 j* k* x*
B5
*x *
x* m* l* x* B5 k* j* x*
18
x x
x* i* h* x* g* f* x* i* h* x* y x A3 f g x d e x f g B3 x x* h* i* x* f* g* x* h* i* x* y
y x d e x b c x d e x y x* x* f* g* g* f* x* x* d* B2 e* A2 e* d* x* x* f* g* g* f* x* x*
y x* i* h* x* g* f* x* i* h* x* B3 g f
A3 y x* h* i* x* f* g* x* h* i* x*
x*
x h i x f g x h i x x* j* k* k* * A4 x B4 j* h* x* i* i* x* j* h* k* * x* x k* j* x*
y y
x* m* l* x* k* j* x* x B5 k j x i h x k j A5 x y x* l* m* x* j* k* x*
x*
x k
h*
x*
k*
j*
x*
x y
x*
l*
* x* j* k* x*
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S5.3
Quantitative amplication gel

10 nM 60 nM 70 nM
70
40 nM
50
20 nM
30 nM
Signal (arbitrary units)
0 0 10 20 30 40 60 I (nM)
Figure S17. Agarose gel electrophoresis demonstrating quantitative amplication. Top: different concentrations of initiator incubated with all hairpin species (A1, A2, B2, 91 nM; the concentration doubles for each subsequent generation of hairpins). The gel measures uorescence emission from Cy5, which is used to label hairpin A1. D denotes dendrimers; M denotes monomers. Bottom: Linear t between the uorescence signal of the dominant reaction product versus the concentration of initiator. Data from three independent experiments are denoted respectively by blue crosses, red diamonds, and green circles. Each set of data is normalized by the signal obtained at 70 nM initiator concentration.
Fig. S17 demonstrates that the concentration of dendrimer depends linearly on the concentration of the initiator in the system.
0n M
50 nM
1n M
5n M
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S5.4
AFM image analysis
~8 nm
I
A1
~16 nm
30 nm 30 nm
A3 B3
B3
A2
B2 A3
35.2 nm
8.6 nm
16.9 nm
28.9 nm
16.8 nm
7.8 nm
G3
B4
A4
I
A1
B4
A4
A3 A2 B3 B2
B3
33.1 nm
14.1 nm
30 nm
30 nm
17.7 nm
7.7 nm
10.5 nm
13.5 nm
A3
A4
B4
A4
B4
33.6 nm
16.5 nm
10.8 nm
21.0 nm
12.8 nm
19.9 nm
G4
8.8 nm 9.6 nm 33.2 nm
Figure S18. AFM measurements of the G3/G4 dendrimers. The small images are screenshots of the measurement section les. The distance between the two red arrowheads is listed above the image. The blue arrows point to the 4-arm junction in both the schematic and the images and help to relate the images to the schematic.
Using a B-DNA model where one helical turn contains 10.5 base pairs and measures 3.4 nm, we calculate the expected arm length for the duplex formed by A1 and I to be 25 / 10.5 3.4 nm = 8.1 nm and the approximate length of all the other duplex segments to be 50 / 10.5 3.4 nm = 16.2 nm. Fig. S18 shows the image analysis for G3 and G4 dendrimers; Fig. S19 shows the image analysis for G5 dendrimers. The measured lengths of the arms are roughly consistent with the calculated lengths. Fig. S20 shows a large eld-of-view AFM image of the G5 system. Note that in most AFM images, only the duplex portions of the dendrimer are visible.
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A5
B5 A5
A5
B5
I
A1
~16 nm
B5 B4 A4 B3 A5
B5
B4 A4 A3 B3
A2
B2
A3 A4 B4 B5
A5 A4 B4 B5 B5 A5 A5
B5
G5
A5
30 nm
30 nm
16.4 nm
17.7 nm
16.9 nm
19.7 nm
35.5 nm
17.8 nm
18.0 nm
28.4 nm
16.9 nm
18.7 nm
18.7 nm
19.7 nm
18.0 nm
18.0 nm
18.0 nm
18.8 nm
17.4 nm
20.7 nm
16.7 nm
17.7 nm
17.8 nm
32.9 nm
19.1 nm
8.2 nm
16.7 nm
16.9 nm
19.0 nm
10.9 nm
7.7 nm
Figure S19. AFM measurements of the G5 dendrimers. The distance between the two red arrowheads is listed above the image. The blue
arrows point to the 5-arm/4-arm junctions in both the schematic and the images and help to relate the images to the schematic. Note that the duplex AI is not visible for the image in the left panel, likely due to damage during sample preparation or AFM scanning.
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100 nm
Figure S20. Large-eld-of-view AFM image of the G5 dendrimer system.
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S6
S6.1
Autonomous locomotion
I B I
A 1
A 2
A 3
A 4
A 5 I B I
Step 1
I I I B B A A 1 A 2 A 3 A 4 A 5 B I 1 A 2
I B
A 1 B
A 2 I
A 3
A 4
A 5
A 3 I B
A 4
A 5 I
A 1
A 2 I
A 3
A 4
A 5 B
A 1
A 2
A 3
A 4
A 5
A 1 2
A 3
A 4
A 5
A 1
A 2 I
A 3
A 4 I
A 5
Step 2
B I B I B A A A A A B I B A A A A I B B B I B A B A A A A B I A
B A A A A
I B A A A
I B A A
A I B B A B A A A A I
Step 3
I B I B A B I B A B B
B A A A A B B A B A A A A B A A A A I I B
B I B A B I
Step 4
B B A I B A B B B A A A A I A A B B B
I B A A B A B B B A A B A A B
I B A B B A A A A
I B
Figure S21. Execution of the reaction graph for the autonomous walker system of Fig. 5. Reaction steps corresponding to the processive
sub-population of walkers are shown in purple. In the initial conditions prior to Step 1, the input ports of the A nodes at sites 1 and 2 are bound to the output ports of the I nodes on the bipedal walker. Execution begins with an assembly reaction between the accessible output port on either of these A nodes and the accessible input port on B. In the top route of Step 1, B assembles with A at site 1, resulting in the disassembly of the trailing I from A, which is then free to assemble with A at site 3, moving the walker one step down the track and bringing the system to Step 2. Alternatively, a B node could bind to A at site 2 prior to the assembly of I with A at site 3, resulting in the disassembly of the walker from the track. The walker could then diffuse through the bulk solution and re-attach to the same track or another track at any A monomer that has not yet been occupied. In the bottom route of Step 1, node B assembles with node A at site 2, resulting in the disassembly of the leading I from A. Due to geometric constraints (inextensible walker torso and rigid track backbone), the walker cannot attach to site 1 and site 3 simultaneously and hence will eventually detach from the track when a B node assembles with A at site 1. Similarly, in Step 2 and Step 3, processive stepping occurs stochastically for a sub-population of walkers. In Step 4, the walker will disassemble from track.
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Fig. S21 depicts the step-by-step execution of the reaction graph for the walker. The initial bond between the output port of I and the input port of A indicates that an assembly reaction has already occurred prior to the execution of the reaction graph. As noted in Sect. S2, static structural elements can impose geometrical constraints on the execution of the reaction graph. In the reaction graph depicted here, the gray structural elements represent a rigid track backbone and an inextensible walker torso; their relative dimensions imply that when one I node is attached to an A node on the track, the other I node can only interact with the A node to either side.
S6.2
Secondary structure of the walker system

y* c y d a x
c*
B
x* b* c* y* y* d* a* x*
Walker
5 nt spacer d* a* 26 bp 5nt spacer x*
b* a* d* y y* c* b* x*
x* x y y* Site 1 A c c* b b* x x* a a* 3nt spacer 15nt
Site 2
c b x a
Site 3
Site 4
Site 5
27nt 71nt
15nt
27nt
15nt
27nt 128nt
15nt
27nt
15nt
15nt
Track
Figure S22. Secondary structure schematic of the walker system of Fig. 5. Stars represent uorophores; black dots represent quenchers. The
lengths of segments a, b, c, and d are 7 nt; the lengths of segments x and y are 2 nt. For sequences, see Sect. S8.6.
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S6.3
Step 1
Step 2
Step 3
Step 4
Figure S23. Step-by-step secondary structure schematic for the autonomous walker system of Fig. 5. Reaction arrows corresponding to
the processive sub-population of walkers are shown in purple.
Fig. S23 depicts the step-by-step secondary structure schematic corresponding to the reaction graph of Fig. S21. A more detailed view of Step 1 is shown in Fig. S24.
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x* x* b* c* y* b* c* y* d* a* x x* y* y c* c b* b x* x a* a a* x* y c b x a y* c* b* x* a* x* y c b x a y* c* b* x* a* x* y c b x a y* c* b* x*
d*
d*
d*
d* a* x* y c b x a
x y* c* b* x* a*
c y d a x y* d* a* x*
y*
c*
y*
c*
b*
b*
x* c b* y* y c* d* y* d a* a d* x* x a* b* x* c* y* y c b x a x y* c* b* x* a*
x* x* b* c* y* d* a* x x* y* y c* c b* b x* x a* a b* c* y*
a* x* y c b x a
d* x* y* c* b* x* y c b x a
a*
a*
d* y* c* b* x*
d* a* x* y c b x a
x* c b* y y* c* y* d* a* d* d a x* a* x x y* c* b* x* a* x y* c* b* x* a* x* y* c* y c b x a b* x* y c b x a
a*
a*
a*
d* y* c* b* x*
b* x* c* y* d* a* x* y c b x a
a* x* c b* y c* y* d x a x y* c* b* x* a* d* y*
x* b* c* y* d* a* x* x y y* c c* b b* x* x a* a
a* x* y c b x a
a*
d* x* y* c* b* x*
a* y c b x a
d* y* c* b* x*
x* b* c* y* d* a* x* x y y* c c* b b* x x* a* a
a* b* x* x* c* b* c* y* y* d* x a* x* y c b x a
c y d a x y* c* b* x* a* d* y*
a* x* y c b x a
a*
a*
d* y* c* b* x*
y*
c*
y*
c*
b*
b* x y* c* b* x* a* x
y* c* b* x* a*
x* b* c*
y* d* a* x* x y y* c c* b b* x x* a a*
x* b* c* y* d* a* x x* y y* c c* b* b x x* a* a
a* x* y c b x a
a*
d* y* c* b* x*
a* x* y c b x a
a*
a*
d* y* c* b* x*
Figure S24. Detailed secondary structure schematic for Step 1 of Fig. S23. Reaction arrows corresponding to the processive sub-population
of walkers are shown in purple.
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S6.4
Assembly of the walker system
The walker system was assembled in four steps (Fig. S25a). Step 0. The walker (W) was assembled by annealing strands W1-BHQ1 and W2-BHQ1 as follows: heat the mixture at 95 C for 5 minutes and slowly cool to room temperature over the course of 4 hours. Step 1. Hairpins S1 and S4 were mixed with track strands S2, S3, and S5, then annealed to produce Track 1 (T1) as follows: heat the system at 95 C for 5 minutes; slowly cool to room temperature over the course of 4 hours. Step 2. T1 and the pre-assembled walker (W) were incubated at room temperature for 2 hours to produce T1+W. Step 3. Hairpins S6, S9, and S11 were mixed with track strands S7, S8, S10, and S12, then annealed to produce Track 2 (T2) as follows: heat the system to 95 C for 5 minutes; slowly cool to room temperature over the course of 4 hours. For the bipedal and monopedal landing control experiments (Fig. S32), the S7 track strand is replaced by S7truncated (see Fig. S40b) so that T1 and T2 remain disjoint. Step 4. T2 and T1+W were incubated at room temperature for 3 hours to produce the nal system, T1+W+T2. Native agarose gel electrophoresis demonstrates a band shifting pattern that conrms on a step-by-step basis the correct assembly of the walker system. (Fig. S25b).
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a
Walker (W) S3 S2 S5 BHQ-1
Torso
BHQ-1
Step 1 S1 S4 S1 S3 S4 S5 Step 2 S2 Track 1 (T1) Track 1 + Walker (T1 + W) JOE S8 S7 TAMRA S10 FAM S12 Step 3 S6 S8 S9 S10 S11 S12 Step 4 S7 Track 2 (T2) S6 S9 S11
S1
S3 S2
S4
S5
S6
S8
S9
S10
S11
S12
S7 Track 1 + Walker +Track2 (T1+W+T2)
Step 1
Step 2 S1 W T1 S6 S2 W S3 S4 S5 (T1)
Step 3 S6 S6 S6 S7 S7 S7 S8 S8 S9 S6 S7 S8 S9 S10 S6 S7 S8 S9 S10 S11
Step 4 S6 T1 S7 W S8 T2 S9 S10 S11 S12 T1+W+T2 (T2)
S1 S1 S1 S1 S2 S2 S2 S3 S3 S4
Figure S25. Assembly of the walker system. a, Assembly procedure. b, Native agarose gel electrophoresis demonstrating the expected
assembly of the system. Samples were annealed and assembled in reaction buffer (4 mM MgCl2 , 15 mM KCl, and 10 mM Tris-HCl, pH = 8.0) with all species at 0.5 M. A 3% native agarose gel was prepared in 1 LB buffer (Faster Better Media, LLC). Samples were loaded with 2 SYBR Gold stain (Invitrogen) and 10% glycerol. The gel was run at 200 V for 40 minutes at room temperature and visualized using an FLA-5100 imaging system (Fuji Photo Film Co., Ltd.)
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S6.5
Characterization of the fuel system
Initiator
I a* x* b* c* y* x
Catalytic formation of duplex A.B a x b c y x* a* I.A d* (1) I a* x* b* c* y* x c* (2) c y d a x B y* d* a* x* y* c* b*
Metastable monomers x* a x b c y a* A x* b* c* y* c y d a x B y* d* a* x* y* d*
A x* b* c* y*
a x b c y x* a* d* y* c* b* x* a* x* b* c* y* x
b* A.B a x b c y x* a* d* y* c* b* x*
y* d* a* x* b* c* y* x a d y c
5 I
+0.5
+0.2
A+B
+0.1
+1
Step 2 Step 1
A+I A
A+B
A+B
A+B
A+B
Figure S26. Fuel system for the walker system. a, Reaction schematic. Hairpins A and B co-exist metastably in the absence of catalyst I.
Catalyst I catalyzes A and B to form duplex AB. Step 1: the toehold a* of I nucleates at the toehold a of A, resulting in the opening of the hairpin A and the formation of the product IA. Step 2: IA, with c* newly exposed, opens hairpin B; B subsequently displaces I from A, producing waste product AB. b, Agarose gel electrophoresis demonstrates catalytic formation of the DNA duplex. The hairpins were prepared in reaction buffer (4 mM MgCl2 , 15 mM KCl, and 10 mM Tris-HCl, pH = 8.0) using a snap-cooling procedure: heating at 90 C for 5 minutes and cooling on ice for 1 minute. The hairpins were allowed to equilibrate at room temperature for 30 minutes before use. Lanes 1-3: A gel shifting assay validates each reaction step depicted in panel (a). Lanes 3-7: Effects of different concentrations of I (1, 0.5, 0.25, 0.1, and 0) on the formation of AB. Reactants were incubated at 1 M at room temperature for 2 hours. Lane 8: AB annealed over 2.5 hours (1 M hairpin species heated at 95 C for 5 minutes and cooled to room temperature over 2.5 hrs). Upon completion of the reaction, the samples were loaded with 5 SYBR Gold stain (Invitrogen) and 10% glycerol into a 2% native agarose gel, prepared with 1 LB buffer (Faster Better Media, LLC). The gel was run at 350 V for 10 minutes at room temperature and visualized using an FLA-5100 imaging system (Fuji Photo Film Co., Ltd.).
Fig. S26 describes the fuel system that powers the walker system. Here, hairpins A and B co-exist metastably in the absence of catalyst I. Catalyst I catalyzes A and B to form duplex AB. Native gel electrophoresis (Fig. S26b) conrms that the hairpins assemble slowly in the absence of the initiator (Lane 7) and that the assembly is dramatically accelerated by the addition of initiator (Lane 3). Disassembly of the initiator enables catalytic turnover as indicated by the nearly complete consumption of hairpins even at sub-stiochiometric initiator concentrations (Lanes 4-6). Catalyst recovery was further investigated using a uorescence quenching experiment (Fig. S27). An initiator labeled with uorophore FAM (6-carboxyuorescein) is incubated with hairpin A. The hybridization of I with A results in the quenching of the FAM signal, presumably due to hybridization induced proximity of FAM to the guanine near the 5 end of hairpin A.5 Introduction of hairpin B releases I from A and hence leads to uorescence signal recovery (Fig. S27a). The observed near-complete recovery of the uorescence signal (after dilution correction) conrms the near-complete recovery of the catalyst (Fig. S27b).
A+B (ann eale d)
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a
I-FAM Quenched
5
A I-FAM.A
B I-FAM
A .B
12
10
11
Dilution correction
Photon counts
10
0.5
1.5
2.5
3.5
Time (hr)
were obtained using a uorometer from Photon Technology International equipped with a temperature controller set at 21 C. A 1.7 mL QS quartz cuvette (Hellma GmbH & Co. KG) was used. Excitation and emission wavelengths were set at 492 nm and 517 nm, respectively, with 4 nm bandwidths. Hairpin species A and B were each snap-cooled in the reaction buffer (4 mM MgCl2 , 15 mM KCl, and 10 mM Tris-HCl, pH = 8.0) by heating at 90 C for 90 seconds and cooling at room temperature for 30 minutes before use. The reaction concentrations of I-FAM (I labeled with a uorophore FAM) and A were 20 nM with B at 40 nM. After recording the baseline signal produced by the catalyst, I-FAM, hairpin A was introduced and resulted in uorescence signal quenching. After the signal plateaued, hairpin B was introduced and near-complete signal recovery (after dilution correction) was observed.
Figure S27. Fluorescence quenching experiment demonstrating catalyst recovery. a, Experimental design. b, Fluorescence data. The data
S6.6
Raw data for the uorescence quenching experiments
Fig. S28 and Fig. S29 present the raw data and curve tting results for the uorescence quenching experiments measuring the proximity of the quenchers (black dots) on the walker feet to the uorophores (colored stars) decorating the track. In Fig. S28, the walker track is decorated with uorophores JOE TAMRA FAM; in Fig. S29, the walker track is decorated with uorophores TAMRA JOE FAM. For each dye ordering, six pairs of experiments were performed (each box contains data for one bipedal and one monopedal experiment that were performed simultaneously in separate cuvettes using the same instrument). Since the walkers motion is not synchronized, the time scale associated with the quenching of a given dye is characterized by approximating the minimum of the corresponding bulk uorescence signal. To mitigate the effect of noise on estimating the location of the minimum, tted double exponential curves (solid) were used to determine the time at which the minimum uorescence (i.e., maximum quenching) was observed (dashed vertical line) for each uorophore. For each curve t, the data points of the initial baseline and those after the point of inection are excluded (as depicted). The same time window was used for tting all data for each pair of boxed experiments (i.e. for all six traces: 3 bipedal and 3 monopedal). All curve ts have an R2 of 0.94 or better.
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Bipedal
JOE TAMRA FAM
1 2 3 4 5 1 2
Monopedal
JOE TAMRA FAM
3 4 5 1 2
Bipedal
JOE TAMRA FAM
3 4 5 1 2
Monopedal
JOE TAMRA FAM
3 4 5 1 2
Bipedal
JOE TAMRA FAM
3 4 5 1 2
Monopedal
JOE TAMRA FAM
3 4 5
10
Photon counts
4 9 8.5 8
10
4 9 8.5 8
10
4 9 8.5 8 1 2 0
10
4 9.5
10
4 9.5
10
9 8.5 8 0 10 4 1 2
8.5 1 10 4 6.5 6 1 10 5 1.7 1.6 1.5 1 2 0 1 2 2 5.5 0 10 5 1 2 2 0 10 4 1 2
8.5 0 10 6.5 6 5.5 0 10 1.7 1.6 1.5 0 1 2 5 1 2 4 1 2
0 10 6.5 6 4
0 10 6.5 6
Photon counts
6.5 6 5.5 0 10 5 1.7
6.5 6 1 10 5 1.7 1.6 1.5 1 2 0 2 5.5 0
5.5 0 10 1.7 5
5.5 0
Photon counts
1.7 1.6 1.5
1.5 0 1 2
1.5 0 1 2
Time (hr)
Time (hr)
Time (hr)
Time (hr)
Time (hr)
Time (hr)
Bipedal
JOE TAMRA FAM
1 2 3 4 5 1 2
Monopedal
JOE TAMRA FAM
3 4 5 1 2
Bipedal
JOE TAMRA FAM
3 4 5 1 2
Monopedal
JOE TAMRA FAM
3 4 5 1 2
Bipedal
JOE TAMRA FAM
3 4 5 1 2
Monopedal
JOE TAMRA FAM
3 4 5
10
Photon counts
4 9 8.5 8 1 2 0
10
10
10
4 9
10
4 9 8.5 1 2 8 0
10
9 8.5 8 0 10 4
9 8 0 10 7 6.5 6 1 10 5 1.6 2 0 10 5 1 2 4
9 8 0 10 7 6.5 6 0 10 5 1 2 4
8.5 8 1 2 0 10 6.6 6.2 5.8 0 10 1.7 5 4
1 10 4
10 6.6 6.2 1 2 5.8 0 10 1.7
Photon counts
6.5 6 5.5 0 10 5 1 2
6.5 6 5.5 0
1 5
Photon counts
1.6 1.4 0
1.6 1.4 0
1.6 1.5 1.5 1 2 0 1 2
1.4 1 2 0 1 2
1.4 0 1 2
Time (hr)
Time (hr)
Time (hr)
Time (hr)
Time (hr)
Time (hr)
Figure S28. Fluorescence data for track with uorophores JOE TAMRA FAM.
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Bipedal
TAMRA JOE FAM
1 2 3 4 5 1
Monopedal
TAMRA JOE FAM
2 3 4 5 1 2
Bipedal
TAMRA JOE FAM
3 4 5 1
Monopedal
TAMRA JOE FAM
2 3 4 5 1 2
Bipedal
TAMRA JOE FAM
3 4 5 1
Monopedal
TAMRA JOE FAM
2 3 4 5
Photon counts
10 5
104 5.5
10
4 5.5
10
10 5
10
5 4.5 0 10 7.8 7.4 7 0 10 5 1.6 1.5 1 2 1.4 0 1 2 0 10 5 1 2 4
4.5 0 10 8 7.5 7 0 10 5 1.7 1.6 1.5 0 4
5 1 2 4.5 0 10 8 7.5 7 1 2 0 10 1.7 1.6 1.5 0 5 1.65 1.55 1.45 0 1 2 4 1 2 0 10 8.5 8 7.5 0 10 5 4 1 2
5 0 10 8.5 8 7.5 0 10 1.65 1.55 1.45 0 5 4 1 2
4.5 0 10 7.8 7.4 7 4
Photon counts
Photon counts
1.6 1.5 1 2 1.4 0
Time (hr)
Time (hr)
Time (hr)
Time (hr)
Time (hr)
Time (hr)
Bipedal
TAMRA JOE FAM
1 2 3 4 5 1
Monopedal
TAMRA JOE FAM
2 3 4 5 1 2
Bipedal
TAMRA JOE FAM
3 4 5 1
Monopedal
TAMRA JOE FAM
2 3 4 5 1 2
Bipedal
TAMRA JOE FAM
3 4 5 1
Monopedal
TAMRA JOE FAM
2 3 4 5
Photon counts
10 5
4 5
10
4 4.8 4.4 4 1 2 0
10
4 4.8 4.4 1 2 0
10
4 5.5
10
4 5.5
10
4.5 0 10 7.5 7 6.5 0 10 1 2
4.5 0 10 7.5 7 6.5 0 10 1.4 1.3 1.2 0 5
5 1 10 4 8.5 8 7.5 0 10 1.7 1.6 1.5 0 1 2 2 0 10 4 1 2
5 0 10 8.5 8 7.5 0 10 1.7 1.6 1.5 0 1 2 4 1 2
10 7.4 7 6.6 0 10 1.6 1.5 1 2 0
4 7.6 7.2 6.8
Photon counts
0 10 1.6 1.5 5
Photon counts
1.4 1.3 1.2 0
Time (hr)
Time (hr)
Time (hr)
Time (hr)
Time (hr)
Time (hr)
Figure S29. Fluorescence data for track with uorophores TAMRA JOE FAM.
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S6.7
Statistical analysis
For the bipedal walker experiment of Fig. 5c, the uorophore ordering along the track is JOE TAMRA FAM. We wish to assess the statistical signicance of the observation that the time differences between consecutive minima in the three quenching curves are positive (i.e., that tTAMRA tJOE > 0 and tFAM tTAMRA > 0). For the monopedal walker experiments of Fig. 5d with min min min min the same ordering of uorophores along the track, we wish to test the statistical signicance of the observations tJOE tFAM > 0 min min and tTAMRA tJOE > 0. Analogous questions apply to the bipedal and monopedal experiments where the uorophores are min min instead ordered TAMRA JOE FAM along the track (data in Fig. 5f and Fig. S29). For each time gap, we obtain six measurements (x1 , x2 , . . . x6 ; sample size n = 6) from independent experiments (Tables S1 and S2). To avoid making the assumption that the underlying distribution is normal, we employ the distribution-free sign test, which applies to any continuous distribution.6 Our null hypothesis is that the median of these measurements is zero (H0 : = 0); our alternative hypothesis is that the median is positive (Ha : > 0). The test statistic, y, is the number of xi s that exceed 0; for all time gaps in Tables S1 and S2, y = 6 because all measured time differences are positive. Using a one-tailed sign test, the P -value is 0.0156 for all tests. Hence, the null hypothesis can be rejected for each time gap at signicance level = 0.0156. The above sign test analysis is preferred to the more familiar t-test analysis which requires the (unjustied) assumption of an underlying normal distribution. For purposes of comparison, we nonetheless include a t-test analysis (demonstrating that even smaller P -values are achieved under the assumption that the measurements are sampled from a normal distribution). In this case, the null hypothesis is that the mean of these measurements is zero (H0 : = 0); the alternative hypothesis is that the mean is positive (Ha : > 0). The test statistic is t = /(s/ n), where s is the computed standard deviation of the measurements.6 For a one-tailed t-test (with ve degrees of freedom; n 1 = 5), the P -values for all time gaps are shown in Tables S1 and S2. In each case, the P -value is smaller than the one for the corresponding sign test. Hence, the null hypotheses can be rejected with an even more stringent signicance level using the t-test.
Bipedal JOE TMR (sec) TMR FAM (sec) Monopedal FAM JOE (sec) JOE TMR (sec)
x1 515.5 143.0 x1 696.8 144.6
x2 588.3 211.5 x2 730.6 337.0
x3 621.8 135.2 x3 659.1 184.8
x4 590.1 103.3 x4 957.9 74.3
x5 669.2 66.0 x5 636.0 443.6
x6 658.5 287.1 x6 656.4 3.4
Median ( ) 606.0 139.1 Median ( ) 678.0 164.7
Sign stat (y) 6 6 Sign stat (y) 6 6
P -value 0.0156 0.0156 P -value 0.0156 0.0156
Mean () 607.2 157.7 Mean () 722.8 198.0
Std Dev (s) 56.1 79.7 Std Dev (s) 120.0 164.8
t-stat 26.5 4.8 t-stat 14.8 2.9
P -value 0.0000 0.0024 P -value 0.0000 0.0169
Table S1. Measured time differences between minima and statistical analysis for six experiments with bipedal or monopedal walkers on the track
with uorophore ordering: JOE TAMRA FAM. For raw data see Fig. S28.
x1 471.3 178.1 x1 286.4 1092.4 x2 658.9 216.2 x2 427.5 1250.6 x3 553.5 144.0 x3 284.1 1430.4 x4 691.7 143.1 x4 428.0 1573.8 x5 615.6 215.8 x5 542.0 1575.1 x6 462.6 245.6 x6 692.2 799.0 Median ( ) 584.6 197.0 Median ( ) 427.8 1340.5 P -value 0.0156 0.0156 P -value 0.0156 0.0156 t-stat 14.7 11.1 t-stat 6.9 10.4 P -value 0.0000 0.0001 P -value 0.0005 0.0001
Bipedal TMR JOE (sec) JOE FAM (sec) Monopedal TMR FAM (sec) FAM JOE (sec)
Sign stat (y) 6 6 Sign stat (y) 6 6
Mean () 575.6 190.5 Mean () 443.4 1286.9
Std Dev (s) 96.1 42.2 Std Dev (s) 156.3 304.4
Table S2. Measured time differences between minima and statistical analysis for six experiments with bipedal or monopedal walkers on the track
with uorophore ordering: TAMRA JOE FAM. For raw data see Fig. S29.
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S6.8
Comparison of walker time scales
Figs S30 and S31 overlay the tted curves from the six independent bipedal and monopedal walker experiments of Figs S28 and S29. To enable comparison in a single plot, all data is normalized: unity corresponds to the nal baseline uorescence value before adding hairpin B and zero corresponds to the minimum of the tted curve. The time axis is translated so that t = 0 corresponds to the time of the nal baseline data point before adding hairpin B. An upper bound on the variability in the time required to add hairpin B and mix the sample in each experiment is approximately 30 secs, representing the uncertainty in comparing curve ts from different experiments along the same time axis. The variability in the traces for each uorophore is higher in Fig. S31 (TAMRA JOE FAM) than in Fig. S30 (JOE TAMRA FAM). The conclusion drawn from this data, that the time scale to visit any one site with the monopedal walker is longer than the time scale to visit all three sites with the bipedal walker, follows from either data set.
a
Normalized Fluorescence
1 0.8 0.6 0.4 0.2 0 0 0.5 1
b
1.0 0.9
Bipedal
JOE TAMRA FAM
1 2 3 4 5
0.8
Monopedal
0.7
JOE TAMRA FAM

1 2 3 4 5
Time (hr) Normalized Fluorescence

1 0.8 0.6 0.4 0.2 0 0 0.5 1 0.3
Normalized Fluorescence Time (hr)

0 0.5 1
0.6
0.5
0.4
1 0.8 0.6 0.4 0.2 0
0.2
0.1
Time (hr)
0.1 0
0.2
0.4
0.6
0.8
1.2
1.4
Time (hr)
Figure S30. Comparison of time scales for bipedal and monopedal walkers using normalized tted curves from the raw uorescence data
of Fig. S28 with track labeled JOE TAMRA FAM. a, For each uorophore, 12 traces (six for each walker type) are plotted together, demonstrating that the bipedal walker visits each anchorage on a faster time scale than the monopedal walker. b, All 36 traces (18 per walker type) are plotted together to demonstrate that the time scale for the monopedal walker to visit any one of the three anchorages is longer than the time scale of the bipedal walker to visit all three anchorages.
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a
1 0.8 0.6 0.4 0.2 0 0 0.5 1
b
1.0 0.9
Bipedal
TAMRA JOE FAM
1 2 3 4 5
0.8
Monopedal
0.7
TAMRA JOE FAM

1 2 3 4 5
Time (hr) Normalized Fluorescence

1 0.8 0.6 0.4 0.2 0 0 0.5 1 0.3
Normalized Fluorescence Time (hr)

0 0.5 1
0.6
0.5
0.4
1 0.8 0.6 0.4 0.2 0
0.2
0.1
Time (hr)
0.1 0
0.2
0.4
0.6
0.8
1.2
1.4
Time (hr)
Figure S31. Comparison of time scales for bipedal and monopedal walkers using normalized tted curves from the raw uorescence data
of Fig. S29 with track labeled TAMRA JOE FAM. a, For each uorophore, 12 traces (six for each walker type) are plotted together, demonstrating that the bipedal walker visits each anchorage on a faster time scale than the monopedal walker. b, All 36 traces (18 per walker type) are plotted together to demonstrate that the time scale for the monopedal walker to visit any one of the three anchorages is longer than the time scale of the bipedal walker to visit all three anchorages.
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S6.9
Control for walker landing effects
a
Bipedal
TAMRA JOE FAM
1 2 3 4 5 1
Monopedal
TAMRA JOE FAM
2 3 4 5 1 2
Bipedal
TAMRA JOE FAM
3 4 5 1 2
Monopedal
TAMRA JOE FAM
3 4 5
b
1
TAMRA
1 0.8 0.6 0.4 0.2 0 0 1 0 0.8 0.6 0.4 0.2 0
JOE
1 0.8 0.6 0.4 0.2 0 1 0
FAM
Normalized fluorescence
Time (hr)
Time (hr)
Time (hr)
c
x 10
4
TAMRA
1.8 x 10
5
JOE
x 10 3.4
5
FAM
Photon counts
10
1.6 9 0 1 0 1
x 10 9.8
x 10 1.7
3.4
x 10
Photon counts
9.4 1.6 3 9 1.5 0 1 0 1 0 1
x 10 8.2
1.5
x 10
x 10
Photon counts
7.8
1.4
2.6 7.4 0 1 1.3
Time (hr)
Time (hr)
Time (hr)
Figure S32. Comparison of time scales for bipedal and monopedal walkers on the full track and on a disjoint track that requires both walker types to diffuse through solution to land on the track (labeled TAMRAJOEFAM). a, These four types of experimental data are depicted with different colors. Red: Bipedal walker on the full track; purple: monopedal walker on the full track; brown: bipedal walker on the disjoint track; green: monopedal walker on the disjoint track. b, For each of the three sites (3, 4, 5), the time scale for the bipedal disjoint track walker (brown traces) is similar to those for the the monopedal full track walker (purple traces) and the monopedal disjoint track walker (green traces), and slower than the time scale for the bipedal walker on the full track (red traces). c, Raw uorescence data and curve ts for the three pairs of bipedal and monopedal walker experiments on the disjoint track. The protocol for these landing experiments was the same as for the other walker uorescence quenching experiments, with the exception that a disjoint track was pre-assembled as described in Sect. S6.4.
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S7
Discussion
S7.1
Leakage and ligation
Commercially available synthetic single-stranded DNA oligos are impure and contain incorrectly synthesized strands.7 The presence of such erroneous strands contributes to the leakage of our systems. To improve strand purity and hence decrease system leakage, we use the following enzyme-based ligation method to synthesize the hairpin monomers: two constituent segments of a hairpin are synthesized and puried separately and ligated to produce the full hairpin (Fig. S33). For DNA sequence details, see Sect. S8. Signicant reduction of system leakage in the ligation-based system is observed, as compared to the un-ligated system (data not shown). We suggest that the observed error reduction may be attributed to the following two mechanisms. First, longer DNA strands contain more errors than shorter fragments, since the shorter fragments can be puried to a higher purity.7 As such, the two constituent segments contain fewer total errors than the full strand. Second, for T4 ligase mediated ligation of Ha and Hb, the successful ligation depends on the correct juxtaposition of the 5 end of fragment Hb with the 3 end of fragment Ha. This requirement provides an additional error reduction mechanism: DNA segments with errors in the regions adjacent to the nick position are not ligated successfully and are eliminated during the subsequent gel purication.
Ha Ha
+
Hb
P
Ligation
Hb
Figure S33. DNA hairpin synthesis by ligation. The circled P indicates a phosphate group, which is required for ligation by T4 ligase.
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S7.2
Molecular compiler
Manual Computerized
Traditional practice
Target structure
Our current practice

Dynamical function
Our future vision

Dynamical function
Reaction graph Nucleic acid secondary structure
Reaction graph
Motif based molecular implementation
Motif based molecular implementation
Nucleic acid primary sequences
Figure S34. Schematic of the molecular compiler vision. a, Standard practice within the nucleic acid design community. b, Our current practice as described in Methods and in Sect. S3.1. c, Our future vision of an integrated compiler for biomolecular function.
Within the nucleic acid design community, it is common practice (Fig. S34a) to specify a design as a set of one or more static target secondary structures.8 The sequences of the constituent strands are then typically designed911 by optimizing an objective function that captures some combination of afnity and/or specicity for the target structures.12 By contrast, we design the dynamic function encoded in a self-assembly system by programming the reaction pathway of the system. The intended dynamic function is rst specied using a reaction graph. The reaction graph is then implemented in terms of the present hairpin motif, and nally the molecular implementation is encoded in the primary sequences of a set of nucleic acid strands (Fig. S34b, also see Sect. S3.1 for details). As such, the standardized motif and the reaction graph provide layers of abstraction that bridge the description of the dynamic behavior of the system and the set of nucleic acid primary sequences, which implement the target behavior. The translation of the target dynamic function to the reaction graph and the subsequent implementation of the reaction graph using the motif are performed manually in this paper. However, standardization of the reaction graph and the motif suggests the feasibility of automating the current manual process. Following this line of thinking, it may eventually be possible to create a compiler for biomolecular function (Fig. S34c). The compiler would take the desired dynamic function as input, translate it rst to a reaction graph, then to a motif-based molecular implementation, and nally into nucleic acid sequences that encode the intended dynamic function.
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S8
DNA sequences
The DNA sequences for the systems described in the paper are presented both as secondary structure schematics (Fig. S35, Fig. S37, Fig. S38, and Fig. S39) and as text sequences annotated with segment names. Note: For each hairpin sequence X, the two segments that are ligated to produce X are indicated as Xa and Xb. Strand modications are indicated as follows:
5 3 3 5 5 5 5 3 phosphorylation: /5Phos/; Cy5: /3Cy5Sp/; 6-carboxyfluorescein: /36FAM/; 6-carboxyfluorescein: /56FAM/; 6-carboxy-4,5-dichloro-2,7-dimethoxyfluorescein (NHS Ester): /5JOEN/; tetrachlorofluorescein: /5TET/; carboxytetramethylrhodamine (NHS Ester): /5TMRN/; black hole quencher-1: /3BHQ_1/
S8.1
Catalytic 3-arm junction system

A C G T
Figure S35. DNA sequences and secondary structures for the catalytic 3-arm junction system of Fig. 2.
The sequences are also listed below as text sequences annotated with segment names.
A: a-x-b-y-z*-c*-y*-b*-x* GCTTGA-GATGTT-AGGGAG-TAGTGC-TCCAAT-CACAAC-GCACTA-CTCCCT-AACATC Aa: GCTTGAGATGTTAGG Ab: /5Phos/GAGTAGTGCTCCAATCACAACGCACTACTCCCTAACATC B: b-y-c-z-x*-a*-z*-c*-y* AGGGAG-TAGTGC-GTTGTG-ATTGGA-AACATC-TCAAGC-TCCAAT-CACAAC-GCACTA Ba: AGGGAGTAGTGCGTT Bb: /5Phos/GTGATTGGAAACATCTCAAGCTCCAATCACAACGCACTA C: c-z-a-x-y*-b*-x*-a*-z* GTTGTG-ATTGGA-GCTTGA-GATGTT-GCACTA-CTCCCT-AACATC-TCAAGC-TCCAAT Ca: GTTGTGATTGGAGCT Cb: /5Phos/TGAGATGTTGCACTACTCCCTAACATCTCAAGCTCCAAT I: y*-b*-x*-a* GCACTA-CTCCCT-AACATC-TCAAGC
The
schematics are generated by the NUPACK server (www.nupack.org).
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S8.2
Catalytic 4-arm junction system
A Ae
B Be
C Ce
D De
A C G T
Figure S36. DNA sequences and secondary structures for the catalytic 4-arm junction system of Fig. S4.
A: a-w-b-x-y*-c*-x*-b*-w*-q* GCTTGA-GATGTT-AGGGAG-TAGTGC-TCCAAT-CACAAC-GCACTA-CTCCCT-AACATC-AACCACCACCAACCACCC Aa: GCTTGAGATGTTAGGGAGTAGTGCTCCAATCACAACGCACTACTCC Ab: /5Phos/CTAACATCAACCACCACCAACCACCC B: b-x-c-y-z*-d*-y*-c*-x*-r* AGGGAG-TAGTGC-GTTGTG-ATTGGA-ACTCAT-CTACCG-TCCAAT-CACAAC-GCACTA-ACAACACACACAAACCAC Ba: AGGGAGTAGTGCGTTGTGATTGGAACTCATCTACCGTCCAATCAC Bb: /5Phos/AACGCACTAACAACACACACAAACCAC C: c-y-d-z-w*-a*-z*-d*-y*-s* GTTGTG-ATTGGA-CGGTAG-ATGAGT-AACATC-TCAAGC-ACTCAT-CTACCG-TCCAAT-ATCCTTCCCTTCCTCTCC Ca: GTTGTGATTGGACGGTAGATGAGTAACATCTCAAGCACTCATCTAC Cb: /5Phos/CGTCCAATATCCTTCCCTTCCTCTCC D: d-z-a-w-x*-b*-w*-a*-z*-t* CGGTAG-ATGAGT-GCTTGA-GATGTT-GCACTA-CTCCCT-AACATC-TCAAGC-ACTCAT-TCTCTTCTTCTCTTCTTC Da: CGGTAGATGAGTGCTTGAGATGTTGCACTACTCCCTAACATCTCAA Db: /5Phos/GCACTCATTCTCTTCTTCTCTTCTTC I: x*-b*-w*-a*
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GCACTA-CTCCCT-AACATC-TCAAGC Ae: q-w GGGTGGTTGGTGGTGGTT-GATGTT Be: r-x GTGGTTTGTGTGTGTTGT-TAGTGC Ce: s-y GGAGAGGAAGGGAAGGAT-ATTGGA De: t-z GAAGAAGAGAAGAAGAGA-ATGAGT
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S8.3
Autocatalytic system
A C G T A
C I
D
Figure S37. DNA sequences and secondary structures for the autocatalytic system of Fig. 3.
A: x*-v*-b*-y*-u*-c*-a*-x*-y-b-v-x-a ACAACT-GAACAC-GTTAGA-CCACTT-CCATCC-TCGCAA-ATCTCC-ACAACT-AAGTGG-TCTAAC-GTGTTC-AGTTGT-GGAGAT Aa-TET: /5TET/TT-ACAACTGAACACGTTAGACCACTTCCATCCTCGCAAATCTCCACAACTAAGTGGTCTAAC Ab-BHQ1: /5Phos/GTGTTCAGTTGTGGAGAT/3BHQ_1/ B: v*-d*-y*-u*-c*-a*-x*-v*-b*-y*-x-a-c-u-y-b GAACAC-TGCTCT-CCACTT-CCATCC-TCGCAA-ATCTCC-ACAACT-GAACAC-GTTAGA-CCACTT-AGTTGT-GGAGAT-TTGCGA-GGATGG-AAGTGG-TCTAAC Ba: GAACACTGCTCTCCACTTCCATCCTCGCAAATCTCCACAACTGAACACGTTAGACCACTTAGTTGTGGAGATTTGCGA Bb: /5Phos/GGATGGAAGTGGTCTAAC C: c-u-y-d-v-u*-c*-a*-x*-v*-d*-y*-u* TTGCGA-GGATGG-AAGTGG-AGAGCA-GTGTTC-CCATCC-TCGCAA-ATCTCC-ACAACT-GAACAC-TGCTCT-CCACTT-CCATCC Ca: TTGCGAGGATGGAAGTGGAGAGCAGTGTTCCCATCCTCGCAAATCTCCACAACTGAACACTGCTCTCC Cb: /5Phos/ACTTCCATCC D: d-v-x-a-c-u-v*-d*-y*-u*-c*-a*-x*-v*-b*-y* AGAGCA-GTGTTC-AGTTGT-GGAGAT-TTGCGA-GGATGG-GAACAC-TGCTCT-CCACTT-CCATCC-TCGCAA-ATCTCC-ACAACT-GAACAC-GTTAGA-CCACTT Da: AGAGCAGTGTTCAGTTGTGGAGATTTGCGAGGATGGGAACACTGCTCTCCACTTCCATCCTCGCAAATCTCC Db: /5Phos/ACAACTGAACACGTTAGACCACTT I: a*-x*-v*-b*-y* ATCTCC-ACAACT-GAACAC-GTTAGA-CCACTT
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S8.4
Nucleated dendritic growth system
A C G T A1 A2 A3 A4 A5
B2 I
B3
B4
B5
Figure S38. DNA sequences and secondary structures for the nucleated dendritic growth system of Fig. 4.
The sequences are also listed below as text sequences annotated with segment names. A1b-Cy5 (together with A1a) is used to synthesize Cy5 labeled hairpin A1.
A1: a-x-c-b-x-y-x*-d*-e*-x*-b*-c*-x*-d*-e*-x* CAAACTC-TT-ATCTATC-TCTGCCA-TT-TT-AA-TGCAATG-TCACGGT-AA-TGGCAGA-GATAGAT-AA-TGCAATG-TCACGGT-AA A1a: CAAACTCTTATCTATCTCTGCCATTTTAATGCAATGTCACGGTAATGGCAGA A1b: /5Phos/GATAGATAATGCAATGTCACGGTAA A1b-Cy5: /5Phos/GATAGATAATGCAATGTCACGGTAA-TT/3Cy5Sp/ A2: b-x-e-d-x-y-x*-f*-g*-x*-d*-e*-x*-f*-g*-x* TCTGCCA-TT-ACCGTGA-CATTGCA-TT-TT-AA-GCTACAG-GACTACG-AA-TGCAATG-TCACGGT-AA-GCTACAG-GACTACG-AA A2a: TCTGCCATTACCGTGACATTGCATTTTAAGCTACAGGACTACGAATGCAATG A2b: /5Phos/TCACGGTAAGCTACAGGACTACGAA A3: d-x-g-f-x-y-x*-h*-i*-x*-f*-g*-x*-h*-i*-x* CATTGCA-TT-CGTAGTC-CTGTAGC-TT-TT-AA-GTATCAG-ATCGCCG-AA-GCTACAG-GACTACG-AA-GTATCAG-ATCGCCG-AA A3a: CATTGCATTCGTAGTCCTGTAGCTTTTAAGTATCAGATCGCCGAAGCTACAG A3b: /5Phos/GACTACGAAGTATCAGATCGCCGAA A4: f-x-i-h-x-y-x*-j*-k*-x*-h*-i*-x*-j*-k*-x* CTGTAGC-TT-CGGCGAT-CTGATAC-TT-TT-AA-TGACCAA-ACCACCT-AA-GTATCAG-ATCGCCG-AA-TGACCAA-ACCACCT-AA
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A4a: CTGTAGCTTCGGCGATCTGATACTTTTAATGACCAAACCACCTAAGTATCAG A4b: /5Phos/ATCGCCGAATGACCAAACCACCTAA A5: h-x-k-j-x-y-x*-l*-m*-x*-j*-k*-x* CTGATAC-TT-AGGTGGT-TTGGTCA-TT-TT-AA-CTCCACT-CCTACTC-AA-TGACCAA-ACCACCT-AA A5a: CTGATACTTAGGTGGT A5b: /5Phos/TTGGTCATTTTAACTCCACTCCTACTCAATGACCAAACCACCTAA B2: x*-f*-g*-x*-d*-e*-x*-f*-g*-x*-y-x-e-d-x-c AA-GCTACAG-GACTACG-AA-TGCAATG-TCACGGT-AA-GCTACAG-GACTACG-AA-TT-TT-ACCGTGA-CATTGCA-TT-ATCTATC B2a: AAGCTACAGGACTACGAATGCAATG B2b: /5Phos/TCACGGTAAGCTACAGGACTACGAATTTTACCGTGACATTGCATTATCTATC B3: x*-h*-i*-x*-f*-g*-x*-h*-i*-x*-y-x-g-f-x-e AA-GTATCAG-ATCGCCG-AA-GCTACAG-GACTACG-AA-GTATCAG-ATCGCCG-AA-TT-TT-CGTAGTC-CTGTAGC-TT-ACCGTGA B3a: AAGTATCAGATCGCCGAAGCTACAG B3b: /5Phos/GACTACGAAGTATCAGATCGCCGAATTTTCGTAGTCCTGTAGCTTACCGTGA B4: x*-j*-k*-x*-h*-i*-x*-j*-k*-x*-y-x-i-h-x-g AA-TGACCAA-ACCACCT-AA-GTATCAG-ATCGCCG-AA-TGACCAA-ACCACCT-AA-TT-TT-CGGCGAT-CTGATAC-TT-CGTAGTC B4a: AATGACCAAACCACCTAAGTATCAG B4b: /5Phos/ATCGCCGAATGACCAAACCACCTAATTTTCGGCGATCTGATACTTCGTAGTC B5: x*-j*-k*-x*-l*-m*-x*-y-x-k-j-x-i AA-TGACCAA-ACCACCT-AA-CTCCACT-CCTACTC-AA-TT-TT-AGGTGGT-TTGGTCA-TT-CGGCGAT B5a: AATGACCAAACCACCTAACTCCACTCCTACTCAATTTTAGGTGGT B5b: /5Phos/TTGGTCATTCGGCGAT I: x*-b*-c*-x*-a* AA-TGGCAGA-GATAGAT-AA-GAGTTTG
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S8.5
Fuel for the walker system

A T G C
Figure S39. DNA sequences and secondary structures for the fuels for the walker system of Fig. S26 and Fig. S27.
A: a-x-b-c-y-x*-a*-d*-y*-c*-b*-x* AAGTAGT-GA-TTGAGCG-TGATGAA-TG-TC-ACTACTT-CAACTCG-CA-TTCATCA-CGCTCAA-TC Aa: AAGTAGTGATTGAGCGTGATGAATGTCACTACTTCAACTCGCATTCATC Ab: /5Phos/ACGCTCAATC B: c-y-d-a-x-y*-c*-b*-x*-a*-d*-y* TGATGAA-TG-CGAGTTG-AAGTAGT-GA-CA-TTCATCA-CGCTCAA-TC-ACTACTT-CAACTCG-CA Ba: TGATGAATGCGAGTTGAAGTAGTGACATTCATCACGCTCAATCACTACT Bb: /5Phos/TCAACTCGCA I: x-y*-c*-b*-x*-a* GA-CA-TTCATCA-CGCTCAA-TC-ACTACTT I-FAM: GACATTCATCACGCTCAATCACTACTT/36FAM/
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S8.6
Walker system
a
x* b* c* y*
y* c y d a x
c*
B
y* d* a* x* b* 5nt spacer d* a* x* y y* c* b* x* a* d*
26 bp x* x 5nt spacer y y* S1 c c* b W2 b* W1 x x* a a* 3nt spacer S3 27nt 15nt 15nt 71nt
S4
S6 c
b x a
S9
S11
S5
27nt
15nt
S8 27nt
128nt
15nt
S10 27nt
15nt
S1215nt
S2
S7
b
y* c y d a x c*
B
y* d* a* x* b* x*
a* d* y y* c* b* x*
x* b* c* y*
S6 c
b 5nt spacer d* a* x a 15nt
S9
S11
S8 27nt
114nt
15nt
S10 27nt
15nt
26 bp x* x 5nt spacer y y* S1 c c* b W2 b* W1 x x* a a* 3nt spacer S3 27nt 15nt 15nt 71nt
S1215nt
S4
S7truncated
S5
27nt
S2
Figure S40. Secondary structure schematics for the walker system. a, Full track. b, Disjoint track for landing control experiments (Sect. S32).
Blue letters indicate sequence names used in the denitions below. The lengths of segments a, b, c, and d are are 7 nt; the lengths of segments x and y are 2 nt. Stars, uorophores; black dots, quenchers.
Note: Sequence B is the same as that described in Sect. S8.5. W1s is used as a splint strand for ligating strands W1a and W1b to produce W1; W2s is used as a splint strand for ligating strands W2a and W2b to produce W2.
S1: GGTAGTTCTAGGCAGCTGAAGTAGTGATTGAGCGTGATGAATGTCACTACTTCAACTCGCATTCATCACGCTCAATC S1a: GGTAGTTCTAGGCAGCTGAAGTAGTGATTGAGCGT S1b: /5Phos/GATGAATGTCACTACTTCAACTCGCATTCATCACGCTCAATC S2: TCATAGGCACCGTCAGACAGGATAGAGCAGTGCATAGATAGTCATAGCCTTGGACCTGCCTAGAACTACC S3: GTCCAAGGCTATGACTATCTATGCACT
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Step 6. Annealed sample CDA reacts with hairpin B and produces a band that corresponds to product CD and AB (Lane 13), which migrates at about the same speed as the annealed product CD (Lane 10) and the annealed product AB (Lane 6), as expected.
S4.4
System kinetics analysis
Analytical modeling The autocatalytic system is modeled using the following reactions:
1 I + A IA
IA + B AB + C ABC + D CD + A CDA + B
2 I + AB 3 ABC 4 AB + CD 5 CDA 6 CD + AB.
k k k k k
To make the system tractable for analytical treatment, we make the following simplifying assumptions: Assumption 1. The forward reaction rates are all the same: ki = k, for i = 1, . . . , 6. This is based on the fact that all the reactions are strand-displacement reactions mediated by 6-nt toe-holds. Under the experimental conditions, the rate limiting step of the toe-hold mediated reactions is the nucleation step,2 the rate of which is determined primarily by the toe-hold length. Assumption 2. The reactions are irreversible. This approximation is justied by the fact that at 25 C, the equilibrium constants for these reactions (e.g., K1 QIA
dt d[CDA] dt d[AB] dt d[CD] dt
= k[AB][C] k[ABC][D] = k[CD][A] k[CDA][B] = k[ABC][D] + k[CDA][B] k[AB][C] = k[ABC][D] + k[CDA][B] k[CD][A].
We next analyze the initial reaction stage of the system when the hairpin reactant depletion is small. Assumption 4. Initial stage assumption. [A] = [A]0 = [B] = [B]0 = [C] = [C]0 = [D] = [D]0 = c0 .
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W2: GTCCGTTCGGTTAGGATACGAGGCAATCCAGGACATTCATCACGCTCAATCACTACTT W2a: GTCCGTTCGGTTAGGATACGAGGCAATCC W2b: AGGACATTCATCACGCTCAATCACTACTT /BHQ-1/ W2s: CGTGATGAATGTCCTGGATTGCCTCGTATC
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References
1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. Yurke, B., Turbereld, A.J., Mills, A.P., Jr., Simmel, F.C. & Neumann, J.L. A DNA-fuelled molecular machine made of DNA. Nature 406, 605608 (2000). Turbereld, A.J. et al. DNA fuel for free-running nanomachines. Phys. Rev. Lett. 90, 118102 (2003). Bois, J.S. Analysis of interacting nucleic acids in dilute solutions. Phd, California Institute of Technology (2007). Yurke, B. & Mills, Jr., A.P. Using DNA to power nanostructures. Genet. Prog. Evolv. Mach. 4, 111122 (2003). Behlke, M.A., Huang, L., Bogh, L., Rose, S. & Devor, E.J. Fluorescence and uorescence applications. Tech. Rep., Integrated DNA Technologies (2005). Devore, J.L. Probability and Statistics for Engineering and the Sciences (Brooks/Cole, 1991). Behlke, M.A. & Devor, E.J. Chemical synthesis of oligonucleotides. Tech. Rep., Integrated DNA Technologies (2005). Seeman, N.C. DNA in a material world. Nature 421, 427431 (2003). Seeman, N.C. De novo design of sequences for nucleic acid structural engineering. J. Biomol. Struct. Dyn. 8, 57381 (1990). Hofacker, I.L. et al. Fast folding and comparison of RNA secondary structures. Chem. Mon. 125, 167188 (1994). Andronescu, M., Fejes, A.P., Hutter, F., Hoos, H.H. & Condon, A. A new algorithm for RNA secondary structure design. J. Mol. Biol. 336, 607624 (2004). Dirks, R.M., Lin, M., Winfree, E. & Pierce, N.A. Paradigms for computational nucleic acid design. Nucleic Acids Res. 32, 13921403 (2004).
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Peng Yin, Harry M. T. Choi, Colby R. Calvert and Niles A. Pierce - Programming Biomolecular Self-Assembly Pathways

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Vol 451 | 17 January 2008 | doi:10.

Assembly and disassembly reactions a* a* t a b c I (1) Assembly at bt ct at a I a* a* t b * A bt b* c b

Execution of reaction graph

Node A Output port c Output port b (inaccessible) (inaccessible)

Catalytic formation of a DNA duplex

Secondary structure mechanism A I A .B

NATURE | Vol 451 | 17 January 2008

Catalytic formation of three-arm junction I.A I (1) a x b y a* x* b* y*

2008 Nature Publishing Group

NATURE | Vol 451 | 17 January 2008

0 (No initiator) 0.0005 (10 pM initiator) 0.001 0.003 0.005 0.01

Exponential amplification A C.D D

2.4 10% completion A+B A

2 1.6 1.2 log10 [I]

I (1) A1 A2 A3 A4 A5 (2) (3) (4) (5) B2 B3 B4 B5 Leakage G1 G2 G3 G4 G5 G5 w/o I

Initiator Metastable monomers

Nucleated dendritic growth I A1 I (1) I A1 G1 (2) A2 G2 B2 B3 G3 (3) A3 A2 B2 I A1 B3 (4)

Signal (arbitrary units)

2008 Nature Publishing Group

NATURE | Vol 451 | 17 January 2008

Site 1 Site 2 Site 3 Site 4 Site 5 Track

0.0 0 Time (h) 1

2008 Nature Publishing Group

NATURE | Vol 451 | 17 January 2008

desired dynamic system behaviour (Supplementary Information 7.2).

2008 Nature Publishing Group

Programming Biomolecular Self-Assembly Pathways

Nucleated dendritic growth

Reaction graph conventions

|a| = |b| = |c| = |x| = |y| = |z| = 6 nt

(3) Sequence design

Step (3) Sequence design. See Methods in main text.

Execution of the reaction graphs

Catalytic formation of a 4-arm junction

Cataytic formation of a 4-arm junction

Extend arms for AFM imaging AFM imaging A.B.C.D.Ae.Be.Ce.De y* c* x* b* w* q* q Ae q Be r Ce y De z t s

A.B.C.D I.A.B.C I.A.B I.A A

AFM image analysis

Design for the catalytic formation of a k-arm junction

Catalytic formation of a k-arm junction

a k-2 x k-2 a k-1 x k-1 x * k-2 a* k-1 x * k-1

a k-1 x k-1 a k x * k-1 a* k

x k-1 a k-1 x k-2 a k-2

Catalytic formation of 6-arm junction a* 1 x * 1 a* 2 x * 2

Detailed secondary structure mechanism

black dot, quencher.

A.B (4b) C.D d v x

)+C (AB C) (AB C)+ D (CD )

A.B.C A.B.C A.B A.B A .I A A.I

A.B, C.D C.D

(CD )+A (CD A)

System kinetics analysis

2 I + AB 3 ABC 4 AB + CD 5 CDA 6 CD + AB.

ABC AB + CD CDA CD + AB.

Nucleated dendritic growth

Execution of the reaction graph

I (1) A1 A2 A3 A4 A5 (2) (3) (4) (5)

Detailed secondary structure mechanism

Nucleated growth of a binary tree (part I)

Nucleated growth of a binary tree (part II)

Quantitative amplication gel

Signal (arbitrary units)