Nanotechnology Science and Computation Part 8 doc

174 B. Yurke 15. J.S. Shin, N.A. Pierce, A synthetic DNA walker for molecular transport, J. Am. Chem. Soc. 126, 10834 (2004). 16. Y. Chen, S H. Lee, C. Mao, A DNA nanomachine based on a duplex-triplex transition, Angew. Chem. Int. Ed. 43, 5335 (2004). 17. Y. Chen, M. Wang, C. Mao, An autonomous DNA nanomotor powered by a DNA enzyme, Angew. Chem. Int. Ed. 43, 3554 (2004). 18. P. Yin, H. Yan, X.G. Daniell, A.J. Tuberfield, J.H. Reif, A unidirectional DNA walker that moves autonomously along a track, A ngew. Chem. Int. Ed. 43, 4906 (2004). 19. D.C. Lin, B. Yurke, and N.A. Langrana, Mechanical Properties of a reversible, DNA-crosslinked polyacrylamide hydrogel, J. Biomech. Eng. 126, 104 (2004). 20. B. Yurke and A.P. Mills, Jr., Using DNA to power nanostructures, Genet. Pro- gram. Evol. Mach. 4, 111 (2003). 21. A.J. Turberfield, B. Yurke, A.P. Mills, Jr., DNA hybridization catalysts and molecular tweezers, in DNA Based Computers V, DIMACS Series in Discrete Mathematics and Theor etical Computer Science Vol. 54, E. Winfree, D. K. Gif- ford, eds., American Mathematical Society, 2000, pp. 171-182. 22. H.X. Qiu, J.C. Dewan, N.C. Seeman, A DNA decamer with sticky end: The crystal structure of d-CGACGATCGT, J. Mol. Bio. 267, 881 (1997). Nanoscale Molecular Transport by Synthetic DNA Machines ∗ Jong-Shik Shin 1 and Niles A. Pierce 1,2 1 Department of Bioengineering 2 Department of Applied & Computational Mathematics, California Institute of Technology, Pasadena, CA 91125, USA niles@caltech.edu 1 Introduction Biological systems have evolved motor proteins programmed to perform in- tracellular transport powered by ATP hydrolysis [23, 14]. Striding along a microtubule with a hand-over-hand gait and a step size of ≈8 nm [26], kinesin is capable of taking ≈100 steps per second, typically negotiating ≈100 steps before falling off the microtubule [4]. Replicating these performance charac- teristics with a synthetic mimic presents a daunting challenge to molecular engineers pursuing programmable active transport as a means to assembling or probing nanoscale systems. DNA nanomachines have been demonstrated to perform mechanical switch- ing between stable states in response to external stimuli [1, 3, 9, 11, 17, 19, 25, 28], and recently several processive DNA motors have been successfully de- signed and constructed. Sherman and Seeman demonstrated a bipedal walker that locomotes in an inchworm fashion, with one foot trailing the other [16]. Inspired by kinesin, the present work describes a bipedal walker that moves by advancing the trailing foot to the lead at each step [18]. Mimicking the peri- odic structure and directional polarity of a microtubule, we construct a DNA track that allows multiple walkers to haul cargo along the same track with directional specificity. These DNA walkers fail to replicate the autonomous nature of kinesin locomotion because they require the manual sequential ad- dition of auxiliary DNA fuel strands. To demonstrate autonomous locomotion, Yin et al. [27] developed a transport mechanism employing ligation and re- striction enzymes powered by ATP, and Tian et al. [21] and Stojanovic and co-workers [20] have developed walkers powered by RNA-cleaving DNAzymes. In principle, autonomous nanomachines can also be powered by hybridization catalysis of metastable DNA molecules [22]. Relative to the speed, versatility ∗ Adapted with permission (Table 1, Figs 1–3, and associated text) from J. Am. Chem. Soc. 2004, 126, 10834–10835. Copyright 2004 American Chemical Society. 176 J S. Shin, N. Pierce and robustness of kinesin, all of these walkers represent initial primitive steps towards a useful framework for achieving programmable active transport with nanoscale resolution. 2 A DNA Walker with the Gait of Kinesin The walker system has four components (Table 1): a walker (W), a track (T), attachment fuel strands (A), and detachment fuel strands (D). The walker consists of two partially complementary oligonucleotides, with a 20-bp helix joining two single-stranded legs (each 23 bases). The track, constructed of six oligonucleotides, has four protruding single-stranded branches (each 20 bases) separated by 15-bp scaffold helices. Neighboring branches run in opposite directions, so spacing of 1.5 helical turns places all branches on the same side of the track approximately 5 nm apart (Fig. 1). As shown in (Fig. 1), the walker strides along the track under the external control of A and D strands. An A strand specifically anchors the walker to a branch by forming helices with the corresponding leg (18-bp) and branch (17- bp). Single-stranded hinges adjacent to either end of these helices (underlined in Table 1) provide flexibility for adopting different conformations depending on the fuel species that are present. When both legs are bound to the track, the trailing leg is released using a D strand that nucleates with the perfectly complementary A strand at a 10-base overhang and then undergoes a strand displacement reaction [28] to produce duplex waste and free the walker leg for the next step. Sequence selection for these system components represents a multi-objec- tive optimization problem requiring the conditional stability of many different secondary structures depending on the subsets of strand species that are con- sidered. Primary sequence design was performed using automated sequence selection software [5] to minimize sequence symmetry [15] while maximizing the probability [7] of adopting a compound secondary structure involving all the strands in the walker system. Subsequent secondary structure prediction [29] for various pairs of strands revealed a small number of undesirable inter- actions, which were eliminated by slight sequence modifications. To detect walker locomotion, all four branches are end-labeled with spec- trally distinct dyes and the two walker legs are end-labeled with quenchers (Table 1 and Sect. 5) to allow monitoring of the fluorescence changes asso- ciated with each dye. Proper monomeric association between the walker and track is demonstrated by examining intermediates during two forward steps using native gel electrophoresis visualized with two different fluorescent scans (Fig. 2). The major band corresponding to fully assembled track (lane 1) is accompanied by minor bands representing partially formed tracks. These par- tial tracks result from slight discrepancies in stoichiometry between the six Nanoscale Molecular Transport by Synthetic DNA Machines 177 Table 1. DNA sequences for the walker and conveyor systems a a Color use is consistent in all graphics. b For the conveyor experiments, W1 is bi- otinylated on the 5  end to facilitate cargo attachment. c For the walker experiments, a track with four branches is constructed from strands T1–T6. For the conveyor ex- periments, a track with four periodically repeating branches is constructed from strands T1 ∗ and T2–T4. d Strands not depicted in Figs. 1 and 4 Fig. 1. Schematic of walker locomotion. Colored spheres represent dyes (HEX, green; Cy5, purple; FAM, red; Texas Red, blue) and quenchers (BHQ1, orange; IBRQ, black) for detecting walker movement. The diagrams depict: (a) unbound walker, (b) walker attached to T1, (c) walker attached to T1 and T2, (d)walker released from T1 to yield duplex waste 178 J S. Shin, N. Pierce Fig. 2. Native polyacrylamide gel electrophoresis demonstrating specific fuel- mediated association of the walker with the track. This figure is a composite of two images obtained by different fluorescent scans of the same gel, using excitation and emission wavelengths that target either HEX on T1 or FAM on T3 (see Sect. 5) track species and lead to the observation of minor bands during subsequent stages of the experiment. The track does not exhibit nonspecific interactions with the walker (lane 2). Anchoring of the walker and subsequent translation results in less mobile intermediates (lanes 3–6). The band intensities of these intermediates are consistent with the expected position of the walker for both wavelength scans. For HEX detection, there is a reduction in band intensity for lanes 3 and 4 relative to lanes 1, 2, 5 and 6. For FAM detection, there is a reduction in band intensity for lane 6 relative to lanes 1–5. The absence of empty track during the operation demonstrates the processivity of walker movement (i.e., at least one leg stays bound to the track). Real-time monitoring of walker movement was carried out by multiplexed fluorescence quenching measurements (Fig. 3). Traversing the track from one end to the other and back, the fluorescent signal from each branch responds specifically to the addition of cognate fuel strands, illustrating high fidelity in controlling walker movement with nanoscale precision. 3 Hauling Molecular Cargo on a DNA Conveyor A periodic track with the same four branches can be constructed from strands T1 ∗ and T2–T4 of Table 1. Multiple walkers can then operate synchronously on the same track to haul tethered molecular cargo with directional specificity under the control of attachment (A) and detachment (D) fuel strands (Fig. 4). Nanoscale Molecular Transport by Synthetic DNA Machines 179 Fig. 3. Real-time multiplexed fluorescence monitoring. The track was preincubated with equimolar walker and A1 for 4 hr at room temperature. Equimolar amounts of A and D fuel strands were successively added from 100× stocks and mixed by rapid pipetting. A different excitation/emission wavelength pair was used to specifically monitor each dye (see Sect. 5). Fluorescence intensities are normalized by the initial track values. The binary digits at the bottom represent the location of the walker on branches T1–T4 (0 for unbound state, 1 for bound state) To verify the elongated structure of the periodic track, the 3  end of T1 ∗ and the 5  end of T2 (i.e., consecutive ends imbedded in the track helix) were each modified with a thiol group and coupled to maleimide-functionalized gold nanoparticles 1.4 nm in diameter. Transmission electron microscopy images (TEM) of the gold-labeled track confirm the formation of long tracks with linear connectivity (Fig. 5). It is not possible to discern the anticipated non- constant spacing between gold particles (expected from labeling neighboring as opposed to evenly spaced strands). Several factors may contribute: incom- plete labeling efficiency (86%), flexibility in the gold linkers, and flexibility of the track backbone under the low-humidity conditions required for TEM analysis. Consistent with significant backbone flexibility, the linear density of gold particles along the (assumed linear) track axis (0.17 ± 0.04 nm −1 ) is higher than the estimated values of 0.10 nm −1 for B DNA (expected in solution) and 0.13 nm −1 for A DNA (expected at low humidity) [12]. Cargo is tethered to the head of the walker by biotinylating the 5  end of W1 (Table 1), allowing binding of either streptavidin or anti-biotin antibody. The gel mobility of the walker decreases when cargo is bound (Fig. 6), with walkers bound to streptavidin (lane 2) migrating faster than those bound to the heavier antibody (lane 3). 180 J S. Shin, N. Pierce Fig. 4. Operation schematic of a molecular conveyor in which multiple walkers haul streptavidin cargo along a periodic track Nanoscale Molecular Transport by Synthetic DNA Machines 181 Fig. 5. Transmission electron microscopy image of a gold-labeled conveyor track (scale bar = 20 nm) Fig. 6. Native polyacrylamide gel elec- trophoresis demonstrating cargo binding. When bound to a biotinylated walker, the mobility of 60 kDa streptavidin [8] is greater than that of the 150 kDa anti- biotin antibody [6]. Lane 1: walker. Lane 2: walker bound to streptavidin. Lane 3: walker bound to anti-biotin antibody Real-time monitoring of orga- nized cargo transport is achieved via multiplexed fluorescence quenching measurements as for the previous walker experiments. Fig. 7 demon- strates cargo transport in both for- ward and backward modes. After adding walkers to a solution contain- ing pre-assembled periodic tracks, cargo transport is controlled by suc- cessive addition of A and D fuel strands. To investigate the effect of cargo size on attachment and detachment kinetics, apparent rate constants were obtained for the cases of no cargo, streptavidin cargo and anti- biotin antibody cargo. Both attach- ment and detachment processes exhibit double-exponential kinetics (data not shown), with fast and slow phases that are relatively insensitive to cargo size (Table 2). Decreases in the attachment rate of ≈10–20% exhibit a positive correlation with increasing cargo size but no trend is evident in the ≈15% variation in detachment rates. Overall, the cargo appears to have a mild im- pact on the kinetics of walker locomotion. 4 Discussion We have demonstrated a synthetic molecular walker that mimics the bipedal gait of kinesin. The present 5 nm step size is smaller than the 8 nm stride 182 J S. Shin, N. Pierce Fig. 7. Real-time monitoring of organized cargo transport a) forwards or b) back- wards along the track. After pre-assembling the tracks, equimolar walker and A and D fuel strands were added from 100× stocks and mixed by rapid pipetting. A differ- ent excitation/emission wavelength pair was used to specifically monitor each dye (see Sect. 5). Fluorescence intensities are normalized by the initial track values. The binary digits at the bottom represent the location of the walker on branches T1 ∗ , T2–T4 (0 for unbound state, 1 for bound state). Nanoscale Molecular Transport by Synthetic DNA Machines 183 Table 2. Apparent rate constants for cargo transport by the molecular conveyor Attachment (0010→0011) Detachment (0011→0010) Cargo fast phase (×10 −2 s −1 ) slow phase (×10 −3 s −1 ) fast phase (×10 −2 s −1 ) slow phase (×10 −3 s −1 ) None 2.98 5.25 1.75 1.98 Streptavidin 2.70 4.75 1.66 1.94 Anti-biotin antibody 2.36 4.29 2.01 2.18 of kinesin on a microtubule [4], and is tunable by adjusting the design of the track scaffold. Construction of a periodic DNA track allows multiple walkers to function as a molecular conveyor by hauling streptavidin cargo along the track in a prescribed direction. The kinetics of walker locomotion are not dra- matically altered by the protein cargo. Generalization to much longer tracks should be achievable by attaching branches to more rigid substrates such as planar [24] or tubular [10, 13] DNA crystals. However, the significant bottle- neck of manually introducing fuel strands at each step makes this approach impractical and highlights the importance of developing autonomous walker designs that operate without human intervention. As a benchmark for walker performance, consider competing with kinesin in a nanomile foot race. Kinesin would make short work of the distance in ap- proximately two seconds. By comparison, the present non-autonomous walker would require one week and one postdoc (ignoring difficulties of yield and exhaustion, respectively). For molecular engineers, there is still much work to do in catching up to the exquisite designs that have evolved in nature. 5 Methods Oligonucleotide and device preparation. DNA oligonucleotides were syn- thesized, labeled and purified by Integrated DNA Technologies. DNA stock solutions were prepared in TE buffer (10 mM Tris, 1 mM EDTA, pH 8.0) and concentrations were determined at 260 nm using the molecular extinction co- efficients provided by the supplier. For the walker experiments, the track was prepared by equimolar mix- ing of the six track strand species (T1–T6) in TSE buffer (10 mM Tris, 150 mM NaCl, 1 mM EDTA, pH 7.5), followed by incubation for 3 hr at 37 ◦ C. The walker was prepared by the same procedure using the two walker strand species (W1, W2). For the conveyor experiments, the periodic track was prepared by equimo- lar mixing of the four track strand species (T1 ∗ , T2–T4) in TSE buffer, fol- lowed by heating to 80 ◦ C for 5 minutes and then slowly cooling to room temperature over 2 hr. Walkers were assembled by combining equimolar W1 biotinylated at the 5  end with W2 in TSE buffer for 3 hr at 37 ◦ C. Protein [...]... 1 988 7 I.L Hofacker, W Fontana, P.F Stadler, L.S Bonhoeffer, M Tacker, and P Schuster Fast folding and comparison of RNA secondary structures Chemical Monthly, 125:167– 188 , 1994 8 K Hofmann, S.W Wood, C.C Brinton, J.A Montibeller, and F.M Finn Iminobiotin affinity columns and their application to retrieval of streptavidin Proc Natl Acad Sci USA, 77 (8) :4666–46 68, 1 980 9 D.A Leigh, J.K.Y Wong, F Dehez, and. .. 4(5):905–909, 2004 18 J.-S Shin and N.A Pierce A synthetic DNA walker for molecular transport J Am Chem Soc., 126:1 083 4–1 083 5, 2004 19 F.C Simmel and B Yurke A DNA-based molecular device switchable between three distinct mechanical states Appl Phys Lett., 80 (5) :88 3 88 5, 2002 20 M.N Stojanovic Personal communication, 2005 21 Y Tian, Y He, Y Chen, P Yin, and C Mao A DNAzyme that walks processively and autonomously... topology Nature, 415( 686 7):62–5, 2002 26 A Yildiz, M Tomishige, R.D Vale, and P.R Selvin Kinesin walks hand-overhand Science, 303:676–6 78, 2004  188 J.-S Shin, N Pierce 27 P Yin, H Yan, X.G Daniell, A J Turberfield, and J.H Reif A unidirectional DNA walker that moves autonomously along a track Angew Chem Int Ed., 43:4906–4911, 2004 28 B Yurke, A.J Turberfield, A.P Mills, Jr., F.C Simmel, and J.L Neumann A... V Balzani, A Credi, S Silvi, and J.F Stoddart A molecular elevator Science, 303: 184 5– 184 9, 2004 4 S.M Block Kinesin: What gives? Cell, 93:5 8, 19 98 Nanoscale Molecular Transport by Synthetic DNA Machines 187 5 R.M Dirks and N.A Pierce A partition function algorithm for nucleic acid secondary structure including pseudoknots J Comput Chem., 24:1664–1677, 2003 6 E Harlow and D Lane Antibodies: A Laboratory... FAM, 5 38/ 555 nm for HEX, 5 98/ 617 nm for Texas Red, and 6 48/ 6 68 nm for Cy5) For the walker experiments, track (0.5 μM) was preincubated with equimolar walker and A1 in TSE buffer for 4 hr at room temperature Equimolar amounts of A and D fuel strands were successively added from 100× stocks and mixed by rapid pipetting For the conveyor experiments, equimolar amounts of the streptavidinloaded walker and fuel... double strands but also confer a variety of metal-based functions upon DNA Such a novel structural motif in DNA will influence research in areas as diverse as nanobiotechnology, materials science, and computer science References 1 H Cao, K Tanaka, and M Shionoya An alternative base pairing of catecholbearing nucleosides by borate formation Chem Pharm Bull., 48: 1745–17 48, 2000 2 E Meggers, P.L Holland, W.B... Multicomponent Assemblies Including Long DNA and Nanoparticles – An Answer for the Integration Problem? Andreas Wolff, Andrea Csaki, and Wolfgang Fritzsche Institute for Physical High Technology, Albert-Einstein-Strasse 9, 07745 Jena, Germany andreas.wolff@ipht-jena.de andrea.csaki@ipht-jena.de wolfgang.fritzsche@ipht-jena.de 1 Introduction The current focus on the combination of nanotechnology with biotechnology... Fygenseon, and E Winfree Design and characterization of programmable DNA nanotubes J Am Chem Soc., 126(50):16344–16352, 2004 14 M Schliwa and G Woehlke Molecular motors Nature, 422:759–765, 2003 15 N.C Seeman Nucleic acid junctions and lattices J Theor Biol., 99:237–247, 1 982 16 W.B Sherman and N.C Seeman A precisely controlled DNA biped walking device Nano Lett., 4(7):1203–1207, 2004 17 J.-S Shin and N.A... 1× TBE buffer (89 mM Tris, 2 mM EDTA, 89 mM boric acid, pH 8. 0) at 50 V and 4◦ C with 6.7% acrylamide The bands were visualized by fluorescent scanning (Molecular Imager FX Pro Plus, Bio-Rad) using 532 nm excitation and a 555 nm cut-off filter to detect HEX on T1, and 488 nm excitation and a 30 nm bandpass filter centered at 530 nm to detect FAM on T3 For the conveyor system, the binding of protein cargo... Tengeiji, T Kato, N Toyama, and M Shionoya A discrete self-assembled metal array in artificial DNA Science, 299:1212–1213, 2003 7 K Tanaka, A Tengeiji, T Kato, N Toyama, M Shiro, and M Shionoya Efficient incorporation of a copper hydroxypyridone base pair in DNA J Am Chem Soc., 124:12494–124 98, 2002 8 K Tanaka, Y Yamada, and M Shionoya Formation of silver(I)-mediated DNA duplex and triplex through an alternative . molecular elevator. Science, 303: 184 5– 184 9, 2004. 4. S.M. Block. Kinesin: What gives? Cell, 93:5 8, 19 98. Nanoscale Molecular Transport by Synthetic DNA Machines 187 5. R.M. Dirks and N.A. Pierce. A partition. hybridization topology. Nature, 415( 686 7):62–5, 2002. 26. A. Yildiz, M. Tomishige, R.D. Vale, and P.R. Selvin. Kinesin walks hand-over- hand. Science, 303:676–6 78, 2004. 188 J S. Shin, N. Pierce 27. P Appl. Phys. Lett., 80 (5) :88 3 88 5, 2002. 20. M.N. Stojanovic. Personal communication, 2005. 21. Y. Tian, Y. He, Y. Chen, P. Yin, and C. Mao. A DNAzyme that walks pro- cessively and autonomously